Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/springer-journals/mltg-gene-deletion-mitigated-virulence-potential-of-streptococcus-4oG5hKIgp6
- Publisher
- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- eISSN
- 2191-0855
- DOI
- 10.1186/s13568-023-01526-x
- Publisher site
- See Article on Publisher Site
Abstract
the virulence of S. mutans. In fact, glycolytic lactic acid Introduction production triggers the pH drop and creates an acidic Amongst tooth health concerns, the formation of den- salivary environment, which accelerates the deminerali- tal caries (DCs) is the most prevalent chronic oral dis- zation of teeth and promotes caries formation. Thus, to ease in both, children and adults of our modern society withstand such an adverse acidic environment being cre- (Miglani 2019). Shockingly, ~ 90% of school going chil- ated on the human tooth surface, S. mutans elicits certain dren and ~ 100% of old age humans survive with the transcriptional and physiologic modulations to overcome pain and discomfort of dental caries in low socioeco- the acid-damage to its DNA synthesis and metabolic nomic status groups worldwide (Petersen et al. 2005; machinery that is collectively termed the "acid tolerance Schwendicke et al. 2015; Fejerskov and Kidd 2008). DC response (ATR) (Banas and Vickerman 2003; Sztajer et al. is a multifactorial, chronic illness caused by oral micro- 2014; Ahn et al. 2006; Lemos et al. 2005; Guo et al. 2015). biota under non-ideal conditions in the oral cavity. The Notably, PG building blocks construct tough yet flex - genesis of DC encompasses the gradual demineraliza- ible protective cell wall architecture which is prone to tion of hydroxyapatite of tooth surface through metabolic have subtle abortive event either during bio-genesis or products, e.g., organic acids. Organic acids are yielded by remodelling of PGs and can consequent in deleterious cariogenic consortia resident bacteria present on tooth kinks and pits in cell wall. In fact, during bacterial cell surfaces adhered biofilms by utilising available sugars division, NAG-NAM (i.e., NAG: N-acetylglucosamine (Fejerskov and Kidd 2008; Loesche 1986). Surprisingly, and NAM: N-acetylmuramic acid) polysaccharide units a single Gram-positive pathogenic Streptococcal species, (n) fuel the biosynthesis of PGs through two distinct among 49 other species in human oral microbiome, can sets of enzymes: (i) polymerases and (ii) lytic transgly- act as a potential infectious agent to lead infections in cosylases (LTs), recruited to connect and re-model the diverse anatomic spaces, including skin, soft tissue, endo- PG building blocks, respectively, to accommodate the carditis, pneumonia, meningitis, sinusitis, otitis media, next PG unit and associated protein assemblies. Each chorioamnionitis, sepsis, and even death, in different age PG strand remains attached covalently to its neighbour- cohorts (Guevara et al. 2020; Kasper et al., 2021). Owing ing strand via peptide stems linked with NAM saccha- to its excellent intrinsic ability to produce sticky glucans, ride, resulting in a cross-linked, net-like PG layer. This S. mutans is often regarded as the most notorious cari- whole polymer is known as the sacculus that encap- ogenic pathogen in the human oral microbiome (Birlutiu sulates the bacterial cell and maintains the cell shape et al. 2018; Hamada and Slade 1980). Such glucans with ( Jorgenson et al. 2014; Winther et al. 2021; Sassine et al. several surface encapsulating auxiliary proteins surround 2021; Bohrhunter et al. 2021; Yunck et al. 2016). How- the S. mutans and thereby facilitate their colonisation in ever, a certain level of hydrolysis in septal PGs is essen- biofilm on the tooth surface. Additionally, its ability to tial in order to (i) create space for incorporation of generate and tolerate organic acids plays a crucial role in Zaidi et al. AMB Express (2023) 13:19 Page 3 of 15 newly generated PG building-blocks and (ii) facilitate Materials and methods the separation of two daughter cells during cell divi- Declarations sion, mediated by dynamic and transient multi-protein This study was carried out following all the institutional complexes, the elongasome and divisome (Wientjes ethical standards. The research on rats was conducted et al. 1991; Silhavy et al. 2010; Vollmer and Seligman with the approval of "Jawaharlal Nehru Medical College, 2010; Egan et al. 2020). For this, LTs congregate with AMU, Institutional Animal Ethics Committee," registra- other proteins as well as enzymes to form machinery tion no. 401/GO/Re/S/2001/CPCSEA. Besides research on that crumbles this layer of PG to release its constitu- extracted human teeth was performed with the approval of ent polysaccharides such as NAG and NAM (Williams “Dr. Ziauddin Ahmad Dental College AMU”, “AMU ethical et al. 2020; Walter and Meyer 2019; Viala et al. 2004). committee”, registration no {(No. 151/201517/PDFWM- This conversion is the hallmark of LT catalysis. Beyond 2015-2017-UTT-31140 (SAII)}. Each applicable guideline, cell wall regulation, the products generated from LTs whether international, national, and/or institutional, has catalysed reactions can induce potential defence mech- been followed for the use of animals. anisms in bacterial cells, such as β-lactam drugs resist- ance mechanisms in Gram-negative P. aeruginosa and Bacterial stains and culture conditions several Enterobacteriaceae. The β-lactams are targeted S. mutans MTCC497 was procured from the Institute of by penicillin-binding proteins (PBPs) to delay cell-wall Microbial Technology, Chandigarh, India. Cells in each synthesis (Lee et al. 2003, 2001; Pratt 2016). This imi - experiment were grown in Todd-Hewitt (TH) broth tation consequently deactivates PBPs and causes inef- supplemented with 0.3% yeast extract and 1% sucrose fective crosslinking of the PG. Later, such cross-linked (Himedia Labs, Mumbai, India) at 37 °C under aerobic PGs are degraded by LTs. Nevertheless, enabling the conditions. However, Mitis salivarius (MS) broth con- role of LTs intimately relates muropeptide recycling to taining appropriate antibiotic was employed for the antibiotic resistance. Despite their critical role in bac- strain selection during the in-vivo study. Kanamycin terial physiology and resistance against antibiotics, (500 µg/mL) was added to the TH medium, according LTs remain unexplored to be considered as significant to the desired experimental conditions for mutant strain drug targets. Besides, LTs can also induce cytotoxicity selection. Plasmid pet28a was employed to amplify the and NF-B-dependent adverse innate immune responses kanamycin cassette. Horse serum was used at 10% of the in the host (Knilans et al. 2017). Therefore, in the pre - total volume of media for transformation. Competence sent study, we engineered the genome of the S. mutans was induced by adding a synthetic competence stimulat- strain by knocking out the mltG gene (one of the lytic ing peptide (CSP) with a purity of > 95% (the amino acid transglycosylases) to explore whether it is involved in sequence of the peptide is NH2-SGSLSTFFRLFNRS- the bacterial virulence by using ex-situ human and in- FTQALGK-COOH) to the culture. CSP was purchased vivo animal tooth models. from "S" Bio Chem, Kerela, India. A 1 mg/mL stock of Obtained results exhibited a significant decrease in synthetic CSP was prepared by dissolving the peptide in different virulence attributes in the mltG deficient con - Milli Q water, stored at − 20 °C. Table 1 lists the prim- struct (ΔmltG) as compared to its wild-type counterpart ers used in qRT- PCR. All the experiments that have in in-vitro, ex-vivo, and in the oral cavity of experimental been performed in this study were replicated three times Wistar rats. Our results and an in-depth review of pub- biologically. lished literature prompted us to conceive a hypothesis, that can address how the mltG gene governs the activities Construction of the mutant by mltG gene deletion of many proteins and enzymes. The results obtained in Figure 1 demonstrates the scheme we adapted to con- the present study are fascinating and provocative for the struct ΔmltG in the S. mutans strain (MTCC 497). researchers who are exploring strategies to control the Indeed, the mutant was constructed by following an pathogenicity of different bacteria by attenuating a par - insertion-deletion strategy. The primers involved in ticular protein or enzyme present in the complex milieu knockout and to confirm gene deletion were enlisted of indigenous biomolecules. To our best knowledge, such in Table 1. Concisely, mltG-up (927 bp) and mltG-dw comparative and systematic evaluation of mltG induced (983 bp) fragments containing upstream and down- virulence paradigm has never been studied in Gram-pos- stream regions along with some sequences of the mltG itive cariogenic human pathogen S. mutans and there are gene were amplified with mltG-upF and mltG-upR only a few studies which have exhibited its role only in and mltG-dwF and mltG-dwR primers, respectively. cell wall remodelling and PG metabolism (Winther et al. Whereas, PcKan (815 bps) from plasmid pet28a was 2021; Sassine et al. 2021; Bohrhunter et al. 2021; Yunck amplified using KanF and KanR primers (Table 1). et al. 2016). Next, the amplicons underwent restriction digestion Zaidi et al. AMB Express (2023) 13:19 Page 4 of 15 Table 1 Determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of three different antibiotics against wild type and ΔmltG type S. mutans Antibiotics Mode of action Group of antibiotic MIC (µg/mL) Wild type S. mutans ΔmltG S. mutans Vancomycin Inhibits cell-wall synthesis Glycopeptide 312.5 78.0 Gentamycin Inhibits protein synthesis Aminoglycoside 312.5 19.0 Ciprofloxacin It inhibits DNA replication Fluoroquinolones 625.0 312.5 Fig. 1 Illustrates the knockout strategy adopted in the study to delete the mltG gene where, GOI = gene of interest, Kan = kanamycin cassette with Pst1 and Bam H1 and were successively ligated to Influence of mltG deletion on MICs of antimicrobial agents create a mltG-up::PcKan::mltG-dw fragment. Finally, against S. mutans this fragment was transformed into S. mutans with the The broth microdilution method was employed to assess aid of synthetic CSP. Through double-crossover homol - the MICs of the antibiotics acting on different targets as ogous recombination, the PcKan replaced the inter- defined earlier (Zaidi et al. 2020). Two-fold microdilu- nal region of the mltG. Template genomic DNA was tions of antibiotics, viz., gentamycin, vancomycin, and extracted from cultures grown on TH agar plates, as ciprofloxacin, were made in THB (100 µL/well) in 96-well described elsewhere (Senadheera et al. 2005; Zeng et al. microtiter plates, separately. The designated wells were 2006). Incorporation of the Kan marker at the appro- seeded with wild and ΔmltG S. mutans cells (~ 107 CFU/ priate locus was validated through PCR and sequencing mL) for each of the antibiotics separately and kept at (data provided in the Additional file 1: Figs. S1 and S2). 37 °C for 48 h. Separate primer pair for the mltG gene, i.e., mltGF and mltGR, was used with the correctly predicted size from Influence of mltG deletion on the growth dynamics of S. the mutants (1074 bp), which contain a kanamycin cas- mutans sette flanked by some base pairs of the mltG region in Overnight grown wild and ΔmltG S. mutans cells were the mltG deficient strain. Wild-type MTCC 497 chro - added to 50 mL fresh THB and allowed to reach the mosomal DNA was used as a negative control. stationary phase separately. The OD600nm of 1 ml ali - quots was recorded at an interval of 1 h, up to 20 h under Zaidi et al. AMB Express (2023) 13:19 Page 5 of 15 identical conditions, though both the cultures reached coated with gold and visualised under SEM, as described the stationary phase after 16 h (Wen et al. 2015; Miller elsewhere (Misba et al. 2017). et al. 2015; Padfield et al. 2020). Biofilm architecture analysis under scanning electron microscopy Influence of mltG gene deletion on biofilm formation Scanning electron microscopy (SEM) was employed Visualization of biofilm formed by wild and ΔmltG to investigate the disruptions in the biofilm formed by S. mutans strains was carried-out using crystal vio- ΔmltG S. mutans. Briefly, 48 h aged wild and ΔmltG let assay (CV), as described elsewhere (Loo et al. 2000). biofilms on glass-coverslips were fixed with 2.5% glutar - Briefly, ~ 107 CFU/mL of test strains in THB (200 µl) aldehyde and 2% formaldehyde in PBS for 2 h at 4 °C, fol- were added to designated wells of a microtiter plate for lowed by serial dehydration with a series of ethanol (20, 48 h at 37 °C. Next, media containing free cells were 40, 60, 80, and 100%). The dried coverslips were then decanted and wells were dried. The dried biofilms in the coated with gold and visualised under SEM, as described wells were stained with CV (0.1%). Subsequently, the CV elsewhere (Misba et al. 2017). was removed, wells were dried, and 200µL of ethanol was added into each well. Finally, the absorbance of CV was recorded at OD630nm on a 96-well microtiter plate Acidogenicity evaluation of wild and ΔmltG S. mutans reader (iMark Microplate Reader, Bio-Rad, 1681130, The intrinsic ability of wild and ΔmltG S. mutans to pro - USA). duce acid as a metabolic by-product of glycolysis was compared by measuring the pH of cell suspensions, as Adherence potential of wild and ΔmltG types of S. mutans described elsewhere (Valdez et al. 2017; Gregoire et al. on glass surfaces 2007). Briefly, freshly overnight grown wild and ΔmltG Cultures (0.2 mL) of wild and ΔmltG S. mutans were S. mutans cells were pelleted (1000 rpm, 5 min), washed added to 1.8 mL of media containing 1% sucrose in (PBS) and re-suspended in 200 mL of KCl (50 mM) and inclined (30°) tubes and kept at 37 °C for 18 h. Broths MgCl2 (1 mM) mixture, enriched with glucose (55.6 mM) containing the non-adhered cells were collected in sep- at pH 7.2 maintaining the cell-count (~ 107 CFU/mL). arate tubes, and adhered cells were washed cautiously A gradual, time-dependent decline in pH up to a cut- with 0.5 mL of PBS to remove the loosely bound cells. off pH of 3.0 was recorded at different points of time The broths, containing un-adhered cells were decanted, (5–360 min) by using a glass pH electrode and expressed and the PBS washes were collected, centrifuged, and re- as the area under the curve (AUC). suspended in PBS. The bacterial cells adhered to the glass surface were extracted by adding NaOH (0.5 M), cen- Aciduricity assay trifuged, and re-suspended in the same volume of PBS. Similarly, the ability to withstand acidic stress was The bacterial cell density in either of the suspensions was assessed by an acid killing assay, as described by Val- determined by evaluating their turbidity at 600 nm. Total dez et al. 2017. Precisely, an aliquot (~ 107 CFU/mL) of bacterial growth was calculated by summing up the val- 18 h aged wild and ΔmltG S. mutans cells were added ues of turbidity for both adhered and non-adhered bacte- to media containing 1% sucrose and allowed to grow to rial cells. The adherence percentage of wild and ΔmltG S. log phase (OD600nm = 0.5). Next, the cells were pel- mutans was enumerated as (Segal et al. 1985). leted (1000 rpm, 5 min) and washed in PBS vigorously. Turbidity of adhered bacteria The pellets were re-suspended in glycine (0.1 M) buff - Adherance% = × 100 ers (200 mL) maintained at different pH values (2.8, 5.0, Turbidity of total bacteria and 7.0) separately. The acid killing of wild and ΔmltG S. mutans cells was evaluated by spreading the 0.1 mL ali- quot from all experiments on THB plates after incubat- Biofilm architecture analysis under scanning electron ing for zero (T0) and 60 min (T60) for each pH value at microscopy 37 oC and incubating the plates for 48 h. Scanning electron microscopy (SEM) was employed to investigate the disruptions in the biofilm formed by ΔmltG S. mutans. Briefly, 48 h aged wild and ΔmltG Influence of ΔmltG deletion over virulence genes biofilms on glass-coverslips were fixed with 2.5% glutar - expressions of S. mutans by qRT‑PCR analysis aldehyde and 2% formaldehyde in PBS for 2 h at 4 °C, fol- The influence of mltG existence over the transcrip - lowed by serial dehydration with a series of ethanol (20, tional expressions of co-existing virulence genes, 40, 60, 80, and 100%). The dried coverslips were then viz. fruA, gtfC, clpA, spaP, vicA, atpA, ropA, comcD, Zaidi et al. AMB Express (2023) 13:19 Page 6 of 15 ccpA, ftsA, murE, murN and gbpB, in ΔmltG S. mutans amplification. A detailed FCM acquisition method for were assessed by qRT-PCR analysis. Briefly, freshly each sample has been appended in the supporting grown wild and ΔmltG S. mutans cells were taken to information. extract as-well-as purify RNA by using Tri-reagent (Sigma-Aldrich, St-Louis, USA) extraction protocol, Comparative in‑vivo dental biofilm and caries Wistar rat as described by Zaidi et al. 2020. RNA concentrations model were estimated by considering valid absorption ratios of Caries-susceptible Wistar rats aged 12–14 weeks were A260nm/A280nm (UV–Vis, Shimadzu, USA), whereas used to investigate the in-vivo effects of the mltG dele - the integrity of the RNAs was estimated by perform- tion on dental plaque and fissure caries. Two groups ing agarose-gel-electrophoresis. To prepare the tran- (N = 5 per group) of adult Wistar rats were assigned to scription (RT) reaction mixture, a high-capacity cDNA be infected with wild and ΔmltG S. mutans, separately, RT kit (Applied Biosystems, USA) was employed. whereas, third group was kept uninfected. Initially, to 1 μg extracted RNA was taken to make cDNA, which eliminate oral bacteria in teeth, all groups were treated was later stored at − 20 °C. Lastly, using SYBR green with cotton swabs loaded with erythromycin (100 mg/ master mix, quantitative qRT-PCR was conducted by mL). To confirm the absence of co-existing un-exper - Step-One software (Applied Biosystems). The reaction imental S. mutans, saliva from all animals was col- mixture for qRT-PCR contained primers and cDNA lected with sterile cotton swabs and plated on MSB agar at a concentration of 100 ng. For primer design, the plates. To support the implantation of wild and ΔmltG genome sequence AE014133.2 was used (Table 1). The S. mutans, rats in both experimental groups were fed a qRT-PCR cycles include denaturation (10 min, 95 °C, sucrose-enriched (5%) diet ad libitum throughout the n = 1) amplification (n = 40 with 15 s at 95 °C of dena- experiment. On day 4th, the animals in first two groups turation), annealing (30 s, 60 °C) with an extension for were orally inoculated with 200 µL of wild and ΔmltG S. 30 s at 72 °C were performed. 16S rRNA was used to mutans (1.4 × 1010 CFU/mL) suspension onto respective normalise the levels of expression of test genes (Misba animal molar surfaces, once per day for 5 consecutive et al. 2017; Forssten et al. 2010). days to allow oral colonization. 1st Group was inoculated with wild-type, whereas, 2nd group with ΔmltG. On the 11th day, animals were screened for successful infection Flow cytometry‑based comparative analysis being developed in the 1st and 2nd groups by oral swab- of mltG influence over lipid, protein, and DNA bing and plating as described above. After inoculations, synthesis in exponentially growing planktonic all animals were fed with their respective (i.e., sucrose and biofilm‑embedded S. mutans cells enriched and normal) diets for the next 10 days to allow Flow cytometry (FCM) analysis was carried out to esti- the maturation of dental plaque on the animal’s teeth to mate and compare the components of elongasome and a detectable extent. The experiments were ended on the divisome of the wild and ΔmltG S. mutans cells. Both 21st day by sacrificing and extracting the lower jaws of the strains were allowed to grow exponentially (O.D. animals for biofilm and DCs visualization. All jaws were 600 nm = 0.5: log phase). Parallel, 48-h-old wild and de-fleshed and suspended in 3.7% formaldehyde until ΔmltG S. mutans cells embedded in biofilm matrices examined under SEM (Miller et al. 2015; Hasan et al. (elongasome and divisome are nonfunctional) were 2015; Kajfasz et al. 2009). harvested by modest sonication (40 amplitude, 10 pulses of 1 s, Sonics & Materials Inc., USA), centrifuga- tion (5000 rpm, 5 min), and repeated washing in PBS. Comparative ex‑situ biofilm formation on a human tooth The obtained pellets were stained with fluorescent model dyes; (i) fluorescein isothiocyanate (FITC, 1 µg mL-l, The difference in intrinsic ability of wild and knockout 8 h, 4 oC), (ii) Nile-red, (10 µg/mL, 30 min 37◦C) and S. mutans to form biofilm was further investigated by cocktail of mithramycin (100 µg/mL, 10 min, 37 oC) employing ex-situ biofilm formation on human tooth and ethidium-bromide (EtBr, 50 µg/mL, 10 min 37 oC), model. Briefly, human molar teeth were extracted from finally diluted to OD600nm = 0.5 in FCM sheath fluid informed outpatient department patients of Dr. Ziaud- to quantify the proteins, lipid, and DNA contents, din Ahmad Dental College and Hospital (DZADC & respectively. The BD X-20 LSR Fortessa FCM equipped H), Aligarh Muslim University, India. The teeth of inter - with Flowjo10 data analysing software was employed est were washed with H O (6%), then examined under 2 2 in these studies. The FCM was first calibrated to detect a 10 × stereomicroscope to confirm the removal of soft 10,000 cells (events) per sample. Forward scatter (FSC) tissues and stored at 4 °C in a 1% sodium azide solution. and side scatter (SSC) were detected using linear Prior to the experiment, the teeth were surface sterilised Zaidi et al. AMB Express (2023) 13:19 Page 7 of 15 under UV radiation exposure for 4 h. Thus, prepared Results teeth were mounted into autoclaved agar in 6 well plates. Deletion of mltG induced enhancement in antibiotic The wells dedicated to wild and knockout S. mutans were susceptibility in S. mutans treated with THB culture media containing ~ 10 CFU/ The deletion of the mltG gene enhanced the suscep - mL of their respective cells. Whereas, uninfected control tibility of ΔmltG type S. mutans to the antibiotics, viz., teeth were given pristine media at 37ºC and 120 rpm/ vancomycin, gentamicin, and ciprofloxacin, which pri - min. After 72 h of incubation, culture media were dis- marily act as potential inhibitors for the biosynthesis of carded, and teeth were washed with sterilised PBS to bacterial (i) cell-wall, (ii) cytoplasmic proteins, and (iii) remove planktonic bacterial cells. Surface intact biofilms DNA, respectively. Data presented in Table 2 exhibits a on teeth were fixed in 2.5% glutaraldehyde for 6 h at 4ºC. clear-cut decline in the MICs of vancomycin, gentamicin, The teeth were treated with alcohols of different dilu - and ciprofloxacin at 78, 19, and 312.5 µg/mL against the tions (30, 50, 70, and 90%) for dehydration of the biofilm ΔmltG type of S. mutans as compared to the wild type, matrix, and finally were coated with gold film by using which was found to be 312.5, 312.5, and 625.0 µg/mL, sputtering. The SEM images were recorded in the range respectively. The enhanced susceptibility trends in ΔmltG of 500–3000 × magnification by using SEM (JEOL, Japan) S. mutans cells signified cell envelope as the key player in (Forssten et al 2010; Tang et al. 2003). tolerance of wide range of antimicrobials. Statistical analysis Influence of mltG deletion on growth dynamics Statistical analysis was performed by one-way analysis of and survival of S. mutans variance (ANOVA) using the Holm-Sidak method with In determining the impact produced by the loss of the multiple comparisons with the control group (Sigma mltG gene on S. mutans growth rate, we explored the Plot 11.0, USA). The level of statistical significance cho - growing pattern of ΔmltG by growth-curve assay in liquid sen was *p < 0.05, unless otherwise stated. Data were pre- medium. The results in Fig. 2A demonstrate that deletion sented in as averaged values of at least three independent of the mltG gene could reduce the growth rate of ΔmltG experiments done in triplicate. S. mutans significantly (p 0.001) at different points of time as compared to the wild-type strain. With respect to the wild type, a significant (p 0.001) reduction in the survival of ΔmltG S. mutans was also estimated as 68.2 ± 4.5% (Fig. 2B). Besides, log phases, i.e., 3–10 h and 5–12 h, Table 2 List of primers (with sequences) used in cloning, confirmation of mltG gene deletion and qRT-PCR analyses Function Primer name Forward sequence (5′ → 3′) Reverse sequence (3′ → 5′) Cloning mltG-upTAC TCG AGA TGC TGT CTA TGT CTT TATG ATC TGC AGC ATA ACA GTT CGT GTT TCTT mltG-dwATG GAT CCG ATG ATC TTT ATT TTG TAGCC ATG TCG ACT ATT TTC TAC GTC TAC GCTA KanATA GGA GGG ATT TAT ATG AGC CAT ATT CAA TTA GAA GAA CTC ATC CAT GGA TGT CTG GAG Transformation mltGATA CTC GAG ATG AAA AAG GCT AAG CAATC ATT GGA TCC TTA CGA ATC ACT GCT TGA analysis qRT-PCR fruAAGC AGA TCA AAC TAC AGA GCC TAC TGGGA CTG CTC GCA CCA TCA gtfCGGT TTA ACG TCA AAA TTA GCT GTA TTAGC CTC AAC CAA CCG CCA CTG TT clpATTT TGG GAG GCC TGT TGC TTGG CAA CGG AGG CAA TAA TC spaPGAC TTT GGT AAT GGT TAT GCA TCA ATTT GTA TCA GCC GGA TCA AGTG vicTGA CAC GAT TAC AGC CTT TGATG CGT CTA GTT CTG GTA ACA TTA AGT CCAAT atpATCT GCC CGA GAA AGA TCG AGAC CAT TGT TGC GGA TTC G atpCTGG AAT GGG ATC GGA CTT TTT TCC AAC CCA CGT AAT TCA AAGG ropAGCG GTC GCT AAT GCT GAA ATCAC GTT GGA CCT CAT CAT GAA comCDACA ATT CCT TGA GTT CCA TCC AAG TGG TCT GCT GCC TGT TGC ccpAGCC AAC TCA TCC TCA GCA ACA GCG CAG CGT GTC ATT AAT TC ftsXGCA GCA AGA TTT GGA TTG GCT TGG GTT GGT CTT ATT murETGC CGA CCA GCC ACA TGA TTTC AGG CAG CAG CAA CTG CAT TTTC murNGAG GCT GGC AAT CAT AAA CGC AGA GAA TAC AGT GAT AA gbpBATG GCG GTT ATG GAC ACG TTTTT GGC CAC CTT GAA CAC CT Zaidi et al. AMB Express (2023) 13:19 Page 8 of 15 Fig. 2 A Growth curves of ΔmltG type (indicated with blue line) and wild type (indicated with grey line) strains of S. mutans. B Survival rate of ΔmltG type with respect to the wild-type has been shown with bar-graph, a significant (p < 0.001) reduction in survival of ΔmltG S. mutans was also estimated as 68.2 ± 4.5%. C Glycolytic curve. Eec ff t of deletion of mltG gene on acidogenicity of S. mutans. Glycolytic acid production was determined by monitoring the pH decrease in glucose solution (1% v/v) over a period of 6 h. Wild-type represented with grey line and ΔmltG type with blue line. D Eec ff t of deletion of mltG gene on aciduricity of S. mutans. Acid tolerance was determined by measuring the survival rate of S. mutans at different pH values such as 2.8, 5.0, and 7.0 on Todd Hewitt agar plates incubated for 48 h at 37˚C. Wild-type represented with grey line and ΔmltG type with blue line. Number of colonies survived expressed in cfu/mL. E Biofilm formation by ΔmltG type (represented with blue bar) and wild-type (represented with grey bar) strains of S. mutans quantified by crystal violet staining. Results are expressed as means ± standard deviations of triplicate assays from two independent experiments. Image inserts show SEM analysis of of biofilm formed by wild and ΔmltG type strains of S. mutans. Scale bar = 10 μm. F Quantitative real time qRT-PCR analysis of specific genes to figure out differentially expressed genes in ΔmltG type. Housekeeping gene 16S rRNA were used for normalization. The expression level of the wild-type is set to one for each gene. Significantly up regulated than wild-type (P < 0.05). Significantly down regulated than wild-type (P < 0.05). The assays were performed in triplicate and the means ± SD from three independent experiments were calculated were considered to estimate the Td of wild and ΔmltG glycolysis, a sugar metabolic pathway, by measuring the types of S. mutans cells, respectively, by fitting an expo - glycolytic and fermentative enzymatic efficiencies up to nential equation (Y = aebx). Precisely, we observed that 6 h under identical experimental conditions. The data in mltG deficiency greatly enhanced the Td of the mltG type Fig. 2C indicate significant acid production with a 0.78-unit to 2.72 h (R2 = 0.962) as compared to the wild S. mutans pH drop in the case of wild-type S. mutans as compared cells at 1.48 h (R2 = 0.965). Growth was observed up to to ΔmltG S. mutans (p < 0.001) after 6 h. This decline trend 20 h, though both types of bacterial cells, i.e., the parental in pH values reflected a tight-coupled relationship between and the mutant types, reached the stationary phase after mltG gene and enzymatic activity of glycolytic enzymes 16 h. These results indicate a critical involvement of the of bacteria. (Hasan et al. 2014; Ban et al. 2012). In fact, a mltG gene not only in cell-wall synthesis but also in several drop in pH value indicates the mitigated aciduric potential growth-promoting and cell division-associated events. as well. Nevertheless, in parallel, we also observed a sig- nificant (p < 0.001) decrease in CFU in both wild type and Glycolytic pH drop and acid killing assay ΔmltG S. mutans cells, as the pH was shifted from alkaline Considering the fact, we strived to explore the influence of to an acidic range i.e., pH 7.0 > , 5.0 > , and > 2.8; however, the presence and absence of mltG gene in S. mutans over Zaidi et al. AMB Express (2023) 13:19 Page 9 of 15 the effect was more pronounced in the constructed mutant confirmed the involvement of mlgG in carbohydrate and type than that of the wild type (Fig. 2D). Besides, 100% protein metabolism, respectively (Kajfasz et al. 2009; Cai elimination of mutant type at acidic pH 2.8 when exposed et al. 2012). for 60 min indicates a significantly (p < 0.001) reduced aci- duricity of ΔmltG S. mutans cells. Taken together, the Influence of mltG deletion over elongasome and divisome trends obtained from the production and tolerance of components (lipid, protein, and DNA) syntheses acidic stress, the role of mltG can be envisioned as hav- in planktonic and biofilm‑embedded S. mutans cells ing involvement in various metabolic pathways that offer by flow cytometry‑based studies a prolonged acid tolerance and survival to aggravate The median values obtained using flow-cytometry anal - pathogenesis. ysis data (Fig. 3B-ii and E-ii) indicated that, compared to wild type, in log-phase growth and biofilm stage, the Loss of mltG impairs the propensity of S. mutans to adhere, synthesis of DNA was down-regulated by 762 and 393 form biofilm and dysregulate the expression of critical median units, respectively (Fig. 3G). Similarly, the dele- genes tion of mltG reduced the protein (5818 median units) Biofilm-like phenotypes formed by the isogenic strains and lipid (364 median units) in log-phase metabolically were quantified and compared. To estimate the variance active S. mutans cells (Fig. 3G), whereas in the case of in the biofilm formation between two different strains, a biofilm, wild, and mltG, an increase in protein (416 crystal violet assay was carried out. ΔmltG exhibited 40% median units) and lipid (695 median units) was observed reduced biofilm formation as compared to the wild type (Fig. 3G). Overall, these results indicate that mltG gene after 48 h of growth, indicating their reduced propensity to deletion influenced the nexus of several biomolecules form biofilm (Fig. 2E). Also, 52% less tendency for adher- involved in elongasome and divisome functions during ence of the ΔmltG type with respect to the wild type was cell morphogenesis. seen. The outcome of the mltG deficiency on S. mutans biofilm architecture was also evident when assessed In‑vivo caries and ex‑situ dental plaque formation through scanning electron microscopy (SEM) (image assessment inserts in Fig. 2E). qRT-PCR was performed to comprehend The formation of bacterial biofilm, plaque, and caries in the effect produced by mltG on the expression level of viru - the presence of natural body conditions such as body lence genes, viz. fruA, gtfC, clpA, spaP, vicA, atpA, ropA, temperature and salivary pH in the oral cavity offer a comcD, ccpA, ftsA, murE, murN, and gbpB. qRT-PCR- complex bacteria-dentin interaction landscape. Never- based evaluation of the transcriptional expression of bio- theless, in rat’s oral cavity experiments, a prolonged expo- film integrity-strengthening genes found fruA, gtfC, gbpB, sure of wild and ΔmltG S. mutans and sugar enriched and comcD in ΔmltG S. mutans to be down-regulated at diet intake could create distinct levels of dentin lesions 0.77 ± 0.04, 0.15 ± 0.01, 0.81 ± 0.02, and 0.91 ± 0.07-fold, and plaque formation. Concisely, the SEM analysis of the respectively, compared to wild-type (1.0-fold) (Fig. 2F). rats’ molar teeth clearly depicted the demineralization of The results evidently supported the involvement of mltG the enamel surface of teeth with caries progression up to gene in structural integrity of biofilm matrices through the dentin layer, thereby indicating the formation of well- bio-synthesis of (i) glucan, (ii) glucan-binding protein syn- established caries in the group infected with wild-type thesis, (iii) quorum sensing inducer molecules and (iv), sur- (Fig. 4Ai). The surface has an evident biofilm embedded face adhesion promotion proteins (Krzyściak et al. 2014; in the EPS pool (Fig. 5Aii–iv), whereas the group treated Senadheera et al. 2005; Senadheera et al. 2007; Ahn et al. with ΔmltG S. mutans exhibited comparatively shallow 2008). In particular, comCD is involved in quorum sensing, caries and depleted biofilm matrix formation, as demon - while gbpB, despite being called a glucan-binding protein, strated in (Fig. 5Bi–iv), respectively. Overall, SEM based has a primary role in cell-wall metabolism. Similarly, the DCs and biofilm observations were in good agreement down-regulation of ftsX, murE, and murN genes in ΔmltG with mltG dependent multifaceted virulence promot- S. mutans as 0.84 ± 0.02; 0.46 ± 0.02; and 0.20 ± 0.06-fold ing activities of S. mutans obtained from the systematic (Fig. 2F), respectively, indicates a significant level of impair - comparative in-vitro studies. Colony forming unit (CFU) ment in cell division-promoting sub-cellular events and were also performed to estimate the proportion of wild thus delays the Td to 2.72 h, compared to the wild-type type and ΔmltG S. mutans on 11th and 21st day on rat’s (Td = 1.48 h), as described above (Senadheera et al. 2005; teeth provided in the Additional file 1: Fig. S3. Ahn et al. 2008; Liu et al. 2014); Besides, down-regulation of the ccpA and clpA genes in mutant S. mutans cells has Zaidi et al. AMB Express (2023) 13:19 Page 10 of 15 Fig. 3 Protein, DNA and Lipid content of wild-type and mltG-mutant were assessed through flow cytometry and represented through histogram overlay in A–C (log phase metabolic cells), and panels D–F (biofilm embedded cells) respectively, where panels i & ii represent the wild-type and mutant-type, respectively. Panel G exhibits median values of the flow cytometry data presented in panels A–F. The TEM micrographs of wild (H) and mltG (I) type S. mutans demonstrate difference in size and shape Fig. 4 Scanning electron micrographs of extracted human teeth. SEM analysis of extracted human teeth to evaluate the level of caries developed by wild-type and ΔmltG-type Zaidi et al. AMB Express (2023) 13:19 Page 11 of 15 Fig. 5 Scanning electron micrographs of aseptically removed rat teeth. SEM analysis of rat’s teeth to evaluate the level of caries developed by wild-type and ΔmltG-type exhibited an augmented sensitivity to antibiotics such as Discussion vancomycin, gentamycin, and ciprofloxacin as compared Endolytic murein-transglycosylase (mltG) is an inner to the wild type. Enhancement in susceptibility of ΔmltG membrane enzyme that belongs to the class of lytic trans- type against vancomycin, as 78 µg/mL, compared to wild glycosylases. mltG possesses endolytic transglycosylase counterpart as 312.5 µg/mL, indicated that the absence activity; hence, it acts as a terminase to endolytically con- of mltG gene ameliorated the activity of LTs in the cell- trol the PG strand elongation. LTs-mediated PG remodel- wall PGs synthesis and remodelling. Consequently, mltG ling was recently highlighted by Winther et al. 2021, who deficient cells may experience the lack of remodelled or showed the interaction between RNA-binding proteins charged PGs building blocks to develop rigid bacterial EloR and mltG at midcell in S. pneumoniae R6. Whereas, cell envelope to evade the antimicrobial stress during Bohrhunter et al. 2021, have also shown the antagonistic the cell proliferation (Suntharalingam et al. 2009). This potential of mltG against cell-wall synthesis by both PG mltG gene induced disruption in the bio-synthesis of PGs polymerases in E. coli. In fact, the inactivation or dele- building blocks can be speculated to lead a compromised tion of mltG can adversely suppress the requirement of cell-wall structure in ΔmltG S. mutans with increased PBP2b, MreCD, RodA, and RodZ, genes that are involved permeability for antimicrobials and extracellular enti- in S. pneumococcal elongation machinery (Tsui et al. ties influx. Nevertheless, after cell-wall disruption medi - 2016). However, the role of mltG in the virulence of bac- ated vancomycin killing, we envisioned an uninterrupted teria is still an untouched topic, as long as we review the influx of antimicrobials into the cytoplasm of ΔmltG S. available literature. Here we evaluated whether mltG is mutans cells. Surprisingly, antibiotics interfering in pro- involved in regulating the overall physiology and differ - tein and DNA synthesis, such as gentamicin and cipro- ent virulence attributes of S. mutans, which has not been floxacin, could effectively reduce the viability (~ 80–90%) documented hitherto in any bacteria. This study has pre - of ΔmltG S. mutans at MICs of 19 and 312.5 µg/mL, sented in-vitro, in-vivo, and ex-vivo experimental find - compared to wild type MICs of 312.5 and 625.0 µg/mL, ings to demonstrate that loss of ΔmltG resulted in lower respectively (Table 2). Importantly, rapid proliferation virulence potential in comparison to the wild-type strain. and clustering of pathogenic bacteria can play a decisive On the basis of our findings, it can be inferred that mltG role in virulence aggravation in several clinical and bio- gene products modulate susceptibility to antibiotics, medical settings. For instance, demineralization of tooth adherence, biofilm formation, growth rate, acidogenic - surfaces largely depends on the robust metabolic activi- ity, and aciduricity in S. mutans. When the MIC of the ties (e.g., production of EPS and organic acids) of rapidly constructed mutant strain, i.e., ΔmltG was evaluated, it Zaidi et al. AMB Express (2023) 13:19 Page 12 of 15 growing and clustering S. mutans cells, which cover the in Fig. 2E). Considering more than 52% reduced effi - dentin and their ecological niche so as to create dental ciency of ΔmltG S. mutans cells to adhere to glass sur- caries or lesions in depth (Niu et al. 2021). This scenario faces prompted us to explore the association of mltG indeed prompted us to investigate the influence of mltG with transcriptional expressions of co-existing viru- deletion on the (i) growth dynamics, (ii) survival, and lence genes. Genes important for biofilm formation (iii) generation doubling time (Td) of ΔmltG S. mutans like, fruA, gtfC, spaP, comCD, gbpB, genes like clpA and wild-type cells under identical experimental condi- and ccpA involved in protein and carbohydrate metab- tions, i.e., culture media (THB), temperature (37 °C), agi- olism respectively, as well as cell divison genes like tation (120 rpm), and incubation time (0–20 h). In this ftsX, murE, murN were found many folds supressed line, the study by Perez et al. 2018, has demonstrated in the mutant type. However, stress-responsive genes that cell division and PG synthesis in Streptococcus spe- like vicA, ropA, and atpC were found overexpressed cies are orchestrated by the coordinated dynamic move- in ΔmltG, indicating the augmented sensing of envi- ment of essential protein complexes. Hence, the deletion ronmental stress (Fig. 2F). Nevertheless, in view of of mltG can be envisioned to negatively induce the cel- obtained trends from mutant and wild S. mutans, we lular events that are tightly associated with mltG regula- can conclude that our presumption to circumvent the tions in S. mutans cells, which leads to a reduced growth virulence of S. mutants in plaque formation and den- rate. Aciduric nature is another strong feature involved in tal cariogenesis though mltG deletion has provided sig- the virulence of S. mutans. Due to this factor, S. mutans nificant insights which are highly relevant in advanced is able to perform glycolysis at markedly low pH values. state-of-the-arts required in clinical management of S. In view of this fact, they are capable of maintaining pH mutants associated inflations. Indeed, during cell mor - homeostasis across the membrane of the bacterial cell phogenesis, particularly in Streptococcus species, bac- so as to regulate cytoplasmic alkalinity (Xu et al. 2011). terial cells create a protective layer of PGs to envelop uTh s, their suppressed acid-producing capability and the cells to sustain a synchronous synthesis of essential aciduricity may lead to the impaired functioning of an biomolecules, including lipids, proteins, carbohydrates, array of enzymes regulating diverse physiological pro- and nucleic acids, to function as elongasome and divi- cesses such as reduced glycolytic efficacies of microbial some complexes accordingly. Particularly, proteins like cells, dysregulation of cell persistence, obstruction in RNA-binding and Ser/Thr kinase in elongasome coor - IPS (intracellular polysaccharides), and EPS produc- dinated protein complex control cell elongation. But, tion, thereby causing the potential mortality of this cari- several elongasome proteins have been found inter- ogenic bacteria (Hasan et al. 2014). Similarly, the study acting with the LTs homolog mltG (Perez et al. 2018). of Hasan et al. 2014, demonstrated that the inhibition At another end, the divisome triggers the synthesis of of major virulence pathways can trigger the impairment essential components to create a septal disc that facili- of the enzymatic potential of a series of enzymes asso- tates binary fission in bacterial cells. At the same time, ciated with physiological processes (such as glycolysis, divisomes also play a central role in the scaffolding, and intra- and extracellular polysaccharide production) localization, and regulation of PG production across the related to the virulence aggravating vigour of S. mutans. cell envelope to create division zones so as to regulate Nevertheless, in view of our results, the role of the mltG the elongation and division of bacterial cells (Winther gene, particularly in S. mutans is associated dental caries et al. 2021 ; Egan et al. 2020). Flow cytometric analy- virulence control, can be strongly advocated. Considering sis was carried out to measure and compare the com- the fact that, in comparison with cell suspensions, multi- ponents of elongasome and divisome (lipids, proteins component biofilm-embedded S. mutans cells do not and DNA) of log phase- wild type and mltG deficient behave identically when exposed to antimicrobial agents, strain cells (during which elongasome and divisome are we found it interesting to assess mltG gene involvement most active). Since, elongasome and divisome and non- in biofilm formation and surface adherence. functional during biofilm stage, biofilm embedded cells The data in Fig. 2E exhibit a 40% reduced propen- were taken to determine the overall change in median sity for biofilm formation in ΔmltG S. mutans as com - values of lipids, proteins and DNA of the whole cell, so pared to wild-type (p < 0.001). SEM-based parallel as to get the idea that variations in the median values in investigation of biofilm adherence on a glass surface log phase is majorly due the changes in the components also validated our results, demonstrating sparsely dis- of elongasome and divisome (Bjarnsholt et al. 2013). As tributed biofilm architecture produced by ΔmltG S. evident from the results of the flow cytometric analysis, mutans as compared to wild-type cells (image inserts maximum variance in the components between wild Zaidi et al. AMB Express (2023) 13:19 Page 13 of 15 and ΔmltG type was found for log phase cells, thereby against S. mutans associated virulence havoc to consider giving the idea of obstructed elongasome and divisome the mltG gene as a potential therapeutic target. activity in mutant cells due to the altered ratio of their three components (lipids, proteins and DNA). In this Supplementary Information context, Besides, we speculate that deletion of the mltG The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13568- 023- 01526-x. gene in S. mutans leads to improper cell division, which was also validated through suppressed expression of Additional file 1: Fig. S1. Sequence data obtained for wild type strain, cell division genes like ftsX, murE, and murN. Defective when balst was performed for forward and reverse orientations, 99% cell division causes enlargement of cell size, as observed homology was matched with mltG gene of S. mutans. Fig. S2. ΔmltG type type searched for forward orientation: Sequence data obtained for mltG under transmission electron microscopy. TEM-based deficient strain,when balst was performed for forward orientation and size analysis also demonstrated a significant change in reverse orientation, 99% homology was matched with vectors containing diameter as 627–675 nm and 448–554 nm in ΔmltG S. kanamycin casette for selection. Kanamycin cassette replaced the mltG gene by homologous recombination and got incorporated in its place. mutans (Fig. 3I) and wild-type cells (Fig. 3H), respec- Fig. S3. Colony forming unit to check the proportion of wild type and tively. Moreover, in spherically shaped bacteria such ΔmltG S. mutans on rat’s teeth. as S. mutans, inhibition of DNA synthesis leads to the inhibition of cell division and an increase in cell surface Acknowledgements area (Higgins et al. 1974). The ex-situ human tooth and Sahar Zaidi is thankful to Department of biotechnology for the support of fel- lowship. We are also thankful to the Animal House Facility, J.N. Medical College Wistar rat model were employed to assess the influence and University Sophisticated Instrumentation Facility (USIF) centre, Aligarh of mltG over plaque formation and cariogenic poten- Muslim University, Aligarh. India, for animal studies and electron microscope tial, respectively. The SEM analysis of human incisor facilities, respectively. Dr. Anmol Chandele is highly acknowledged for the sup- port in carrying out flow cytometry at ICGEB, Delhi. teeth exposed to wild-type (Fig. 4Bi–iii) and ΔmltG S. mutans (Fig. 4 Ci–iii) evidently supported the ΔmltG Author contributions mediated impairment of biofilm formation on the SZ conceived and designed research. SZ, KA, YC conducted experiments. SZ and KA analysed data. SZ wrote the manuscript. KA revised the manuscript. All teeth’s enamel surface. A clear-cut reduction in ΔmltG authors read and approved the final version of the manuscript. S. mutans density while biofilm formation progressed is in good agreement with our mltG-mediated biofilm Funding This study was supported by internal funds of Interdisciplinary Biotechnology ameliorating trends, as discussed above. Unit and DBT grant: BT/PR40148/BTIS/137/20/2021. The present study demonstrates the systematic, devel - opment of the mltG deficient gene construct in S. mutans Availability of data and materials All data is available as main figures and table in manuscript as well as sup - and the evaluation of the virulence of wild and ΔmltG S. plementary data attached. mutans under three different biofilm models, including (i) in-vitro glass surface adherence and (ii) ex-situ human Declarations tooth and (iii) in-vivo animal molar tooth models for bio- film and cariogenic lesions. Preliminary results showed Ethics approval and consent to participate Each applicable guideline either international, national, and/or institutional that S. mutans virulence-associated activities such as cell have been followed for the use of animals and human teeth. The study is division, Td, acid tolerance, metabolism of sugars to pro- approved by Institutional ethical committee as mentioned in method section. duce organic acids and EPS, and components of divisome Consent for publication and elongasome were significantly deregulated in ΔmltG We have taken consent in written and the same may not be disclosed as per S. mutans as compared to the wild type, particularly for the university policies. log phase cells. Next, perturbation in the transcriptional Competing interests expressions of S. mutans’ genes, which solely contribute No potential conflict of interest was reported by the authors. to biofilm formation, quorum sensing, EPS production, and cell-wall building block synthesis, has warranted the Author details Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary, association of mltG with the nexus of complex metabolic Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, UP, India. systems. Besides, mltG deletion-based mitigation in viru- ICGEB-Emory Vaccine Center, International Centre for Genetic Engineering lence of S. mutans in dental settings, that is, human and and Biotechnology, New Delhi, India. rodent’s dentin lesions demonstrated a clear-cut com- Received: 3 February 2023 Accepted: 7 February 2023 promised tooth infection trend in the case of ΔmltG S. mutans, compared to the wild type counterpart. Overall, these results provided significant insights that can effec - tively encourage the research developments progressing Zaidi et al. AMB Express (2023) 13:19 Page 14 of 15 References Streptococcus mutans. J Bacteriol 191:2060–2068. https:// doi. org/ 10. 1128/ Ahn SJ, Wen ZT, Burne RA (2006) Multilevel control of competence develop-JB. 01609- 08 ment and stress tolerance in Streptococcus mutans UA159. Infect Immun Kaspar JR, Lee K, Richard B, Walker AR, Burne RA (2021) Direct interactions with 74:1631–1642. https:// doi. org/ 10. 1128/ IAI. 74.3. 1631- 1642. 2006 commensal streptococci modify intercellular communication behaviors Ahn SJ, Ahn SJ, Wen ZT, Brady LJ, Burne RA (2008) Characteristics of biofilm for - of Streptococcus mutans. ISME J 15:473–488. https:// doi. org/ 10. 1038/ mation by Streptococcus mutans in the presence of saliva. Infect Immun s41396- 020- 00789-7 76:4259–4268. https:// doi. org/ 10. 1128/ IAI. 00422- 08 Knilans KJ, Hackett KT, Anderson JE, Weng C, Dillard JP, Duncan JA (2017) Ban SH, Kim JE, Pandit S, Jeon JG (2012) Influences of Dryopteris crassirhizoma Neisseria gonorrhoeae lytic transglycosylases LtgA and LtgD reduce extract on the viability, growth and virulence properties of Streptococcus host innate immune signalling through TLR2 and NOD2. ACS Infec Dis mutans. Molecules 17:9231–9244. https:// doi. org/ 10. 3390/ molec ules1 3:624–633. https:// doi. org/ 10. 1021/ acsin fecdis. 6b000 88 70892 31 Krzyściak W, Jurczak A, Kościelniak D, Bystrowska B, Skalniak A (2014) The Banas JA, Vickerman MM (2003) Glucan-binding proteins of the oral strepto- virulence of Streptococcus mutans and the ability to form biofilms. Eur J cocci. Crit Rev Oral Boil Med 14:89–99. https:// doi. org/ 10. 1177/ 15441 Clin Microbiol Infect Dis 33:499–515. https:// doi. org/ 10. 1007/ s10096- 013- 11303 01400 203 1993-7. (Epub 2013 Oct 24) Birlutiu V, Birlutiu RM, Costache VS (2018) Viridans streptococcal infective endo- Lee W, McDonough MA, Kotra LP, Li ZH, Silvaggi NR, Takeda Y (2001) A 1.2 Å carditis associated with fixed orthodontic appliance managed surgically snapshot of the final step of bacterial cell wall biosynthesis. Proc Natl by mitral valve plasty: a case report. Medicine. https:// doi. org/ 10. 1097/ Acad Sci USA 98:1427–1431. https:// doi. org/ 10. 1073/ pnas. 98.4. 1427 MD. 00000 00000 011260 Lee M, Hesek D, Suvorov M, Lee W, Vakulenko S, Mobashery S (2003) A Bjarnsholt T, Ciofu O, Molin S, Givskov M, Høiby N (2013) Applying insights mechanism-based inhibitor targeting the DD-transpeptidase activity of from biofilm biology to drug development—can a new approach be bacterial penicillin-binding proteins. J Am Chem Soc 125:16322–16326. developed? Nat Rev Drug Discov 12:791–808. https:// doi. org/ 10. 1038/ https:// doi. org/ 10. 1021/ ja038 445l nrd40 00 Lemos JA, Abranches J, Burne RA (2005) Responses of cariogenic streptococci Bohrhunter JL, Rohs PD, Torres G, Yunck R, Bernhardt TG (2021) MltG activity to environmental stresses. Curr Issues Mol Biol 7:95–108. https:// doi. org/ antagonizes cell wall synthesis by both types of peptidoglycan polymer-10. 21775/ cimb. 007. 095 ases in Escherichia coli. Mol Microbiol 115:1170–1180. https:// doi. org/ 10. Liu X, Miller P, Basu U, McMullen LM (2014) Sodium chloride-induced filamen- 1111/ mmi. 14660 tation and alternative gene expression of fts, murZ, and gnd in Listeria Cai J, Tong H, Qi F, Dong X (2012) CcpA-dependent carbohydrate catabolite monocytogenes 08–5923 on vacuum-packaged ham. FEMS Microbiol Let repression regulates galactose metabolism in Streptococcus oligofermen- 360:152–156. https:// doi. org/ 10. 1111/ 1574- 6968. 12599 tans. J Bacteriol 194:3824–3832. https:// doi. org/ 10. 1128/ JB. 00156- 12 Loesche WJ (1986) Role of Streptococcus mutans in human dental decay. Egan AJ, Errington J, Vollmer W (2020) Regulation of peptidoglycan synthesis Microbiol Rev 50:353–380. https:// doi. org/ 10. 1128/ mr. 50.4. 353- 380. 1986 and remodelling. Nat Rev Microbiol 18:446–460. https:// doi. org/ 10. 1038/ Loo CY, Corliss DA, Ganeshkumar N (2000) Streptococcus gordonii biofilm s41579- 020- 0366-3 formation: identification of genes that code for biofilm phenotypes. J Fejerskov O, Kidd E (2008) Dental caries: the disease and its clinical manage- Bacterial 182:1374–1382. https:// doi. org/ 10. 1128/ jb. 182.5. 1374- 1382. 2000 ment. Wiley, New Jersey Miglani S (2019) Burden of dental caries in India: Current scenario and future Forssten SD, Björklund M, Ouwehand AC (2010) Streptococcus mutans, caries strategies. Int J Clin Pediatr Dent 13:155. https:// doi. org/ 10. 5005/ jp- journ and simulation models. Nutrients 2:290–298. https:// doi. org/ 10. 3390/ als- 10005- 1733 nu203 0290 Miller JH, Avilés-Reyes A, Scott-Anne K, Gregoire S, Watson GE, Sampson E Gregoire S, Singh AP, Vorsa N, Koo H (2007) Influence of cranberry phenolics (2015) The collagen binding protein Cnm contributes to oral coloniza- on glucan synthesis by glucosyltransferases and Streptococcus mutans tion and cariogenicity of Streptococcus mutans OMZ175. Infect Immun acidogenicity. J Appl Microbiol 103:1960–1968. https:// doi. org/ 10. 1111/j. 83:2001–2010. https:// doi. org/ 10. 1128/ IAI. 03022- 14 1365- 2672. 2007. 03441.x Misba L, Zaidi S, Khan AU (2017) A comparison of antibacterial and antibiofilm Guevara MA, Lu J, Moore RE, Chambers SA, Eastman AJ, Francis JD, Noble efficacy of phenothiazinium dyes between Gram-positive and Gram- KN, Doster RS, Osteen KG, Damo SM, Manning SD (2020) Vitamin D and negative bacterial biofilm. Photodiagnosis Photodyn 18:24–33. https:// Streptococci: the interface of nutrition, host immune response, and anti-doi. org/ 10. 1016/j. pdpdt. 2017. 01. 177 microbial activity in response to infection. ACS Infect Dis 10:3131–3140. Niu JY, Yin IX, Wu WK, Li QL, Mei ML, Chu CH (2021) Antimicrobial peptides for https:// doi. org/ 10. 1021/ acsin fecdis. 0c006 66 the prevention and treatment of dental caries: a concise review. Arch Oral Guo L, McLean JS, Lux R, He X, Shi W (2015) The well-coordinated linkage Biol 122:105022. https:// doi. org/ 10. 1016/j. archo ralbio. 2020. 105022 between acidogenicity and aciduricity via insoluble glucans on the Padfield D, Castledine M, Buckling A (2020) Temperature-dependent changes surface of Streptococcus mutans. Sci Rep 5:1–1. https:// doi. org/ 10. 1038/ to host–parasite interactions alter the thermal performance of a bacterial srep1 8015 host. ISME J 14:389–398. https:// doi. org/ 10. 1101/ 554717 Hamada S, Slade HD (1980) Biology, immunology, and cariogenicity of Strep- Perez AJ, Cesbron Y, Shaw SL, Villicana JB, Tsui HC, Boersma MJ (2018) Move- tococcus mutans. Microbiol Rev 44:331–384. https:// doi. org/ 10. 1128/ mr. ment dynamics of divisome and penicillin-binding proteins (PBPs) in cells 44.2. 331- 384. 1980 of Streptococcus pneumoniae. bioRxiv 1:429217. https:// doi. org/ 10. 1073/ Hasan S, Singh K, Danisuddin M, Verma PK, Khan AU (2014) Inhibition of major pnas. 18160 18116 virulence pathways of Streptococcus mutans by Quercitrin and Deoxyno- Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C (2005) The jirimycin: a synergistic approach of infection control. PLoS ONE 9:e91736. global burden of oral diseases and risks to oral health. Bull World Health https:// doi. org/ 10. 1371/ journ al. pone. 00917 36 Organ 83:661–669. https:// doi. org/ 10. 1371/ journ al. pone. 02688 99 Hasan S, Danishuddin M, Khan AU (2015) Inhibitory effect of zingiber officinale Pratt RF (2016) β-Lactamases: why and how. J Med Chem 59:8207–8220. towards Streptococcus mutans virulence and caries development: in vitro https:// doi. org/ 10. 1021/ acs. jmedc hem. 6b004 48 and in vivo studies. BMC Microbiol 15:1–4. https:// doi. org/ 10. 1186/ Sassine J, Pazos M, Breukink E, Vollmer W (2021) Lytic transglycosylase MltG s12866- 014- 0320-5 cleaves in nascent peptidoglycan and produces short glycan strands. Cell Higgins ML, Daneo-Moore L, Boothby D, Shockman GD (1974) Eec ff t of inhibi- Surf 7:100053. https:// doi. org/ 10. 1016/j. tcsw. 2021. 100053 tion of deoxyribonucleic acid and protein synthesis on the direction of Schwendicke F, Dörfer CE, Schlattmann P, Foster Page L, Thomson WM, Paris cell wall growth in Streptococcus faecalis. J Bacteriol 118:681–692. https:// S (2015) Socioeconomic inequality and caries: a systematic review and doi. org/ 10. 1128/ jb. 118.2. 681- 692. 1974 meta-analysis. J Dent Res 94:10–18. https:// doi. org/ 10. 1177/ 00220 34514 Jorgenson MA, Chen Y, Yahashiri A, Popham DL, Weiss DS (2014) The bacterial 557546 septal ring protein RlpA is a lytic transglycosylase that contributes to Segal R, Pisanty S, Wormser R, Azaz E, Sela MN (1985) Anticariogenic activity rod shape and daughter cell separation in Pseudomonas aeruginosa. Mol of licorice and glycyrrhizine I: inhibition of in vitro plaque formation by Microbiol 93:113–128. https:// doi. org/ 10. 1111/ mmi. 12643 Streptococcus mutans. J Pharm Sci 74:79–81. https:// doi. org/ 10. 1002/ jps. Kajfasz JK, Martinez AR, Rivera-Ramos I, Abranches J, Koo H, Quivey RG Jr 26007 40121 (2009) Role of Clp proteins in expression of virulence properties of Zaidi et al. AMB Express (2023) 13:19 Page 15 of 15 Senadheera MD, Guggenheim B, Spatafora GA, Huang YC, Choi J, Hung DC, Publisher’s Note Treglown JS, Goodman SD, Ellen RP, Cvitkovitch DG (2005) A VicRK signal Springer Nature remains neutral with regard to jurisdictional claims in pub- transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf lished maps and institutional affiliations. expression, biofilm formation, and genetic competence development. J Bact 187:4064–4076. https:// doi. org/ 10. 1128/ JB. 187. 12. 4064- 4076. 2005 Senadheera MD, Lee AW, Hung DC, Spatafora GA, Goodman SD, Cvitkovitch DG (2007) The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance. J Bacteriol 189:1451–1458. https:// doi. org/ 10. 1128/ JB. 01161- 06 Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2:a000414. https:// doi. org/ 10. 1101/ cshpe rspect. a0004 Suntharalingam P, Senadheera MD, Mair RW, Levesque CM, Cvitkovitch DG (2009) The LiaFSR system regulates the cell envelope stress response in Streptococcus mutans. J Bacteriol 191:2973–2984. https:// doi. org/ 10. 1128/ JB. 187. 12. 4064- 4076. 2005 Sztajer H, Szafranski SP, Tomasch J, Reck M, Nimtz M, Rohde M, Wagner-Döbler I (2014) Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans. ISME J 8:2256– 2271. https:// doi. org/ 10. 1038/ ismej. 2014. 73 Tang G, Yip HK, Cutress TW, Samaranayake LP (2003) Artificial mouth model systems and their contribution to caries research: a review. J Dent 31:161–171. https:// doi. org/ 10. 1016/ s0300- 5712(03) 00009-5 Tsui HC, Zheng JJ, Magallon AN, Ryan JD, Yunck R, Rued BE, Bernhardt TG, Win- kler ME (2016) Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39. Mol Microbiol 100:1039–1065. https:// doi. org/ 10. 1111/ mmi. 13366 Valdez RM, Duque C, Caiaffa KS, Dos Santos VR, Loesch ML, Colombo NH (2017) Genotypic diversity and phenotypic traits of Streptococcus mutans isolates and their relation to severity of early childhood caries. BMC Oral Health 17:1–9. https:// doi. org/ 10. 1186/ s12903- 017- 0406-1 Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, Moran AP, Athman R, Mémet S, Huerre MR, Coyle AJ, DiStefano PS (2004) Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 5:1166–1174. https:// doi. org/ 10. 1038/ ni1131 Vollmer W, Seligman SJ (2010) Architecture of peptidoglycan: more data and more models. Trends Microbiol 18:59–66. https:// doi. org/ 10. 1016/j. tim. 2009. 12. 004 Walter A, Mayer C (2019) Peptidoglycan structure, biosynthesis, and dynam- ics during bacterial growth. In: Extracellular sugar-based biopoly- mers matrices, Springer, Cham, pp 237–99. https:// doi. org/ 10. 1007/ 978-3- 030- 12919-4_6 Wen ZT, Bitoun JP, Liao S (2015) PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans. PLoS ONE 10:e0124319. https:// doi. org/ 10. 1371/ journ al. pone. 01243 19 Wientjes FB, Woldringh CL, Nanninga N (1991) Amount of peptidoglycan in cell walls of Gram-negative bacteria. J Bacteriol 173:7684–7691. https:// doi. org/ 10. 1128/ jb. 173. 23. 7684- 7691. 1991 Williams AH, Wheeler R, Deghmane AE, Santecchia I, Schaub RE, Hicham S, Nilges MM, Malosse C, Chamot-Rooke J, Haouz A, Dillard JP (2020) Defec- tive lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis. Elife 9:e51247. https:// doi. org/ 10. 7554/ eLife. 51247 Winther AR, Kjos M, Herigstad ML, Håvarstein LS, Straume D (2021) EloR interacts with the lytic transglycosylase MltG at midcell in Streptococcus pneumoniae R6. J Bacteriol 203:e00691-720. https:// doi. org/ 10. 1128/ JB. 00691- 20 Xu X, Zhou XD, Wu CD (2011) The tea catechin epigallocatechin gallate sup- presses cariogenic virulence factors of Streptococcus mutans. Antimicrob Agents Chemother 55:1229–1236. https:// doi. org/ 10. 1128/ AAC. 01016- 10 Yunck R, Cho H, Bernhardt TG (2016) Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria. Mol Microbiol 99:700–718. https:// doi. org/ 10. 1111/ mmi. 13258 Zaidi S, Singh SL, Khan AU (2020) Exploring antibiofilm potential of bacitracin against Streptococcus mutans. Microb Pathog 149:104279. https:// doi. org/ 10. 1016/j. micpa th. 2020. 104279 Zeng L, Wen ZT, Burne RA (2006) A novel signal transduction system and feed- back loop regulate fructan hydrolase gene expression in Streptococcus mutans. Mol Microbiol 62:187–200. https:// doi. org/ 10. 1111/j. 1365- 2958. 2006. 05359.x
Journal
AMB Express – Springer Journals
Published: Feb 20, 2023
Keywords: S. mutans; mltG; Virulence; Biofilm; Proteins; Dental caries