Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

Edited by John J. Mekalanos, Harvard University, Boston, MA, and approved August 20, 2021 (received for review April 6, 2021)
September 29, 2021
118 (40) e2106555118

Significance

Previous studies have indicated that Gram-positive bacteria are not affected by type VI secretion serum (T6SS) intoxication. However, here we show that Acinetobacter baumannii employs its T6SS to kill different Gram-positive bacteria. Furthermore, we determined that killing was dependent on Tse4, a bifunctional effector possessing lytic transglycosylase and endopeptidase activities. Tse4 represents a broad family of modularly organized T6SS peptidoglycan-degrading effectors. In addition, we show that secretion of D-lysine by A. baumannii results in a pH increase, which greatly enhances Tse4 activity. These results expand the range of T6SS-mediated interbacterial interactions that may shape the composition of bacterial communities in the context of the human microbiota and polymicrobial infections.

Abstract

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.
Bacteria live in dense communities and are often in constant competition with other bacterial species to secure nutrients and space. Bacterial warfare is mediated by the production of diffusible antimicrobial compounds and by sophisticated molecular weapons, such as the type VI secretion system (T6SS). The T6SS is a dynamic nanomachine that delivers toxic effector proteins from an attacking cell (predator) to nearby competitors (prey) in a contact-dependent manner. Although bacteria possess a diverse arsenal of toxic effector proteins to kill Gram-negative bacteria, current evidence suggests that Gram-positive bacteria are immune to T6SS attacks. Immunity to T6SS-dependent killing between Gram-negative kin cells is accomplished by the expression of immunity proteins, which specifically bind and inactivate their cognate effector. Broad-spectrum mechanisms of protection against nonkin T6SS attacks in Gram-negative bacteria have only recently been uncovered (1, 2).
Due to its essentiality, the bacterial cell wall peptidoglycan (PG), also known as murein, is targeted by various T6SS effectors. PG is composed of glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid (MurNAc) that are crosslinked through MurNAc-attached peptides. PG-degrading (PGase) effectors induce bacterial cell lysis, which facilitates the complete clearance of bacterial competitors by preventing dead cells from shielding susceptible prey from T6SS attacks, a phenomenon known as the “corpse barrier” effect (3). The PGase effectors characterized to date contain one lytic enzymatic activity and can be categorized as muramidases, which cleave the β-(1,4)-glycosidic bond (4, 5), N-acetylmuramyl-L-alanine amidases, which unlink the peptides chains from MurNAc (6) and LD (4, 7), or DD-endopeptidases (7), which cleave PG crosslinks.
T6SS-dependent bacterial warfare generates an arms race in which both predator and prey evolve tools to prevail. We recently showed that some Acinetobacter baumannii strains, such as strain ATCC 17978 (Ab17978), use the periplasmic racemase RacK to produce the noncanonical D-amino acid (NCDAA) D-lysine (D-Lys) from L-Lys. Incorporation of D-Lys into the PG of Ab17978 underlies a defensive strategy against the PGase activity of T6SS effectors from competing bacteria (2). Beyond being incorporated into the PG, most of the D-Lys that is produced is secreted into the extracellular milieu (2). It has been recently suggested that NCDAAs carry out diverse biological roles in bacterial ecosystems (8). In this work, we investigate the role of extracellular D-Lys in bacterial warfare.

Results

RacK Enhances Tse4-Mediated T6SS Killing by A. baumannii.

Previous work has shown that T6SS-dependent toxicity can be impacted by the extracellular environment (9). We hypothesized that D-Lys secretion could potentiate T6SS-dependent bacterial killing by Ab17978. To test this hypothesis, we compared the T6SS killing activity of wild-type (WT) Ab17978 with that of its racK deletion (ΔracK) derivative. We found that the T6SS of WT Ab17978 is ∼100 fold more lethal than the ΔracK strain against two different prey, Escherichia coli MG1655 and Acinetobacter nosocomialis M2 with an inactive T6SS (M2ΔtssB) (Fig. 1A). As expected, Ab17978ΔtssM, which possesses an inactive T6SS, did not display bacterial killing (Fig. 1A). WT and ΔracK secreted equal amounts of the T6SS protein Hcp, indicating that deletion of racK does not affect T6SS dynamics (SI Appendix, Fig. S1). We also confirmed that Ab17978ΔtssM secretes similar amounts of D-Lys compared with the WT strain, suggesting that T6SS functionality and D-Lys secretion are independent processes (SI Appendix, Fig. S2). Remarkably, heterologous racK expression in the clinical isolate A. baumannii strain UPAB1, which does not encode racK, resulted in a 100-fold increase in T6SS lethality compared with WT (Fig. 1B). Together, these data demonstrate that D-Lys secretion enhances the T6SS-mediated killing of A. baumannii.
Fig. 1.
RacK enhances T6SS-dependent killing by A. baumannii. (A) Competition assay using Ab17978 WT, ΔracK, or ΔtssM as predators and A. nocosomialis M2ΔtssB or E. coli MG1655 as prey. (B) Competition assay using A. baumannii UPAB1 WT, with UPAB1 heterologously expressing racK (racK+) or racK+ΔtssM as predators and A. nocosomialis M2ΔtssB or E. coli MG1655 as prey. Bar graphs represent means of prey survival CFU after 4 h of coincubation ± SD of at least three biological replicates. Statistical analyses were performed using the unpaired Student’s t test; *P < 0.05, ***P < 0.001.
Ab17978 encodes four T6SS effectors: Tse1 is a predicted lipase; Tse2 is a predicted nuclease; Tse3 is an effector of unknown function; and Tse4 (ACX60_00605) is predicted to contain PGase activity (10). A. nosocomialis M2 does not encode any of these effectors or their cognate immunity proteins. To gain insight into the effectors involved in D-Lys–mediated enhancement of T6SS lethality, we tested the killing efficiency of Ab17978 mutants unable to secrete one or more effectors against M2ΔtssB. We found that a mutant strain unable to secrete effectors Tse1, Tse2, and Tse3 (Δ123) retained most of its killing ability against M2ΔtssB, indicating that Tse4 plays a major role in bacterial killing (Fig. 2). A ΔracK derivative of the Δ123 strain (Δ123ΔracK) lost most of its killing capacity, indicating that RacK enhances Tse4 toxicity. Immunity proteins are generally encoded adjacently to their cognate effector. Indeed, overexpression of tsi4 (ACX60_00610) in M2ΔtssB prey prevented killing by Ab17978Δ123, indicating that Tsi4 is the immunity protein of Tse4 (Fig. 2). Furthermore, the enhanced killing provided by RacK disappeared when M2ΔtssB expressed tsi4 (Fig. 2). Together, our results strongly suggest that the synergistic effect of RacK on T6SS lethality is linked to the cell wall-targeting effector Tse4.
Fig. 2.
Synergistic effect of RacK on bacterial killing is linked to PG hydrolase effector Tse4. Competition assay using Ab17978 WT, ΔracK, ΔtssM, Δ123, and Δ123ΔracK as predators and M2ΔtssB either harboring an empty vector (Left panel) or overexpressing Tsi4, the immunity protein of Tse4 (Right panel) as prey. Bar graphs represent means of prey survival CFU after 4 h of coincubation ± SD of at least three biological replicates. Statistical analyses were performed using the unpaired Student’s t test; *P < 0.05, **P < 0.01, ns = not significant.

Accumulation of Secreted D-Lys Leads to a Local Increase in Environmental pH.

Next, we performed interbacterial competition assays between the Ab17978ΔracK predator and M2ΔtssB prey in media supplemented with increasing amounts of D-Lys. We found that T6SS-dependent prey killing by Ab17978ΔracK was augmented by the extracellular addition of 20 mM D-Lys (SI Appendix, Fig. S3A). Importantly, growth of Ab17978 predator strains and M2 prey strains was not affected by the presence of 20 mM D-Lys in the media (SI Appendix, Fig. S5), thus excluding the possible toxicity of D-Lys alone. Extracellular NCDAAs, such as D-Met, can be incorporated into the PG of nonproducing species (11). To test whether the enhanced T6SS lethality observed in the presence of D-Lys can be emulated by other NCDAAs, we performed bacterial killing assays in the presence of D-Met, D-Ala, or D-Arg. While D-Met and D-Ala did not potentiate T6SS lethality (SI Appendix, Fig. S3 C and D), D-Arg mimicked the effect of D-Lys (SI Appendix, Fig. S3B). Unexpectedly, we found that L-Lys and L-Arg also synergized with the T6SS (SI Appendix, Fig. S3E). These results indicate that basic amino acids, in either their L- or D- form, enhance the lethality of the T6SS. Thus, we then hypothesized that the RacK-dependent enhancement of interbacterial killing was due to a D-Lys–mediated increase in the pH at the interface between WT Ab17978 and its prey. Supporting this concept, when killing assays were performed in a buffered pH of 6.8 (the pH of Luria–Bertani [LB] media), we found that the presence of RacK or 20 mM D-Lys no longer increased the bactericidal effect of Ab17978 (Fig. 3A and SI Appendix, Fig. S4). In contrast, bacterial killing by Ab17978 WT and ΔracK was increased in a buffered pH of 8.0 (Fig. 3A). Employing the pH indicators m-cresol purple and phenol red, we determined that RacK strongly contributes to a local pH increase when Ab17978 is grown in solid growth media (Fig. 3B). Using a standard curve correlating D-Lys concentration to the change in m-cresol purple color, we determined that after 2 h of incubation, the concentration of D-Lys in the microenvironment of Ab17978 WT cells is ∼60 mM (SI Appendix, Fig. S6), nearly three times higher than the maximum concentration of D-Lys employed in the experiment described in SI Appendix, Fig. S3A. The pH of LB media containing 60 mM D-Lys was measured at 9.1.
Fig. 3.
RacK-dependent alkaline environmental conditions enhance bacterial killing and Tse4 activity in vitro. (A) Competition assay using Ab17978 WT, ΔracK, or ΔtssM as predators and M2ΔtssB as prey in nonbuffered media or media buffered at pH 6.8 or 8.0. Bar graphs represent means of prey survival CFU after 4 h of coincubation of four biological replicates. (B) Relative quantification of pH following Ab17978 growth in solid media. Normalized Ab17978 WT, ΔracK, or ΔtssM cultures were spotted on LB agar supplemented with either m-cresol purple (pH = 7.4: yellow, 9.0: purple) or phenol red (pH = 6.2: yellow, 8.2: magenta). (Left) Pictures of agar plates were taken after 2 h of incubation. Colony blot analysis of relative Ab17978 glycan amounts was performed as a control for bacterial growth rate. (Right) Bar graph represents ratio between m-cresol purple pixel signal in grayscale and anti-Ab17978 glycan coly blot IRDye800 signal. Values are mean ± SD of three independent replicates. (C) RBB-labeled sacculi were incubated with 7.5 µM lysozyme or purified Tse4. After ∼16 h, the undigested PG was pelleted, and released dye was quantified by measuring dye absorbance at 585 nm. Values represent mean of three independent enzymatic assays ± SD. Statistical analyses were performed using the unpaired Student’s t test; *P < 0.05, ****P < 0.0001.
Previous work has shown that the pH of the bacterial periplasm (site of Tse4 toxicity) is equal to the pH of the extracellular environment (12). Thus, we hypothesized that the increased T6SS-dependent killing at alkaline pH could be due to enhanced Tse4 activity in this condition. To this end, we tested the in vitro activity of purified Tse4 using a Remazol Brilliant Blue (RBB) dye release assay. This assay measures the soluble products released upon treatment of RBB-labeled insoluble PG with Tse4. We found that the enzymatic activity of Tse4 was optimal at pH 8 and above (Fig. 3C). Based on this data, we propose a model in which D-Lys secretion increases T6SS-mediated killing by creating an alkaline microenvironment in the predator-prey interface, resulting in maximal Tse4 activity. Notably, in Fig. 1B, we showed that adding racK to strain UPAB1 also enhances its T6SS-mediated killing. We hypothesized that UPAB1 also encodes a pH-sensitive PG hydrolase effector. Indeed, we found that effector Tse2 showed a pH-dependency profile similar to Tse4 (SI Appendix, Fig. S7). Furthermore, deletion of tse2 in UPAB1 completely abolished the enhancing effect of racK expression on prey killing (SI Appendix, Fig. S7). These results indicate that basic environmental conditions can enhance the bactericidal effect of various PG-targeting A. baumannii effectors.

RacK and Tse4 Enable T6SS-Dependent Killing of Gram-Positive Bacteria.

The dramatic RacK-induced killing efficiency of Ab17978 prompted us to test if this bacterium is able to kill Gram-positive bacteria. Remarkably, we found that Ab17978 can kill strains of Bacillus subtilis, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in a T6SS- and RacK-dependent manner (Fig. 4A). These Gram-positive preys did not inhibit Ab17978 growth during coincubation (SI Appendix, Fig. S8). Notably, we found that the Δ4 strain, which lacks tse4, did not kill any of these strains, while the Δ123 strain exhibited increased killing in all cases. We hypothesize that the Δ123 strain secretes increased amounts of Tse4 due to the lack of competition for the T6SS machinery, which is consistent with previous work in Pseudomonas aeruginosa (13). Importantly, the addition of purified Tse4 to B. subtilis cultures did not induce cell lysis (Fig. 4B), and coincubation of Ab17978 and B. subtilis in liquid media did not result in bacterial killing (SI Appendix, Fig. S9), indicating that T6SS-dependent activity against Gram-positive bacteria is contact-dependent. Our results are a demonstration of T6SS-dependent killing of Gram-positive bacteria.
Fig. 4.
Ab17978 kills various Gram-positive bacteria in a Tse4- and contact-dependent manner. (A) Competition assay using Ab17978 WT, ΔtssM, ΔracK, Δ4, or Δ123 as predators and B. subtilis JH642 or SCK6, S. aureus USA300 LAC, and L. monocytogenes EDG-e as prey. Bar graphs represent means of prey survival CFU after coincubation ± SD of at least three biological replicates. Statistical analyses were performed using the unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. (B) Late exponential phase growing B. subtilis cells (OD600 ∼1) were treated with either 7.5 µM of lysozyme or Tse4 for 2 h. Survival CFUs were enumerated at 0, 1, and 2 h of treatment. Values represent means ± SD of three biological replicates.

Tse4 Exhibits Both Lytic Transglycosylase and Endopeptidase Activity In Vitro.

Like most Gram-negative bacteria, the PG of B. subtilis and L. monocytogenes use D-Ala-meso diaminopimelate (mDAP) crosslinks (Fig. 5A) (14). In contrast, S. aureus PG uses a penta-Gly bridge connecting the peptide stems (Fig. 5A) (14). Therefore, Tse4 appears to have the remarkable ability to degrade different PG chemotypes. The Conserved Domain Architecture Retrieval Tool algorithm predicted that Tse4 has three conserved domains: a LysM domain in the N-terminal region followed by a lysozyme-like domain and a Zinc-binding peptidase domain. Consistently, the Phyre2 server identified two conserved catalytic domains in Tse4 (SI Appendix, Fig. S10). Amino acid residues 242 to 447 modeled to a lysozyme-like domain that was structurally similar to lytic transglycosylase gp144 of bacteriophage phiKZ (PDB: 3BKH), and this domain would therefore be predicted to nonhydrolytically cleave the glycan chains between N-acetylglucosamine and MurNAc, thus producing anhydromuropeptides. Amino acid residues 634 to 798 modeled to two distinct structures. In the first, the peptidase domain was modeled to the DD-endopeptidase ShyA of Vibrio cholerae (PDB:6U2A), which cleaves D-Ala-mDAP bonds (15). In the second, the peptidase domain was modeled to the lysostaphin LytM of S. aureus (PDB: 2B44), an antibacterial enzyme that is capable of cleaving the penta-Gly bridges found in the PG crosslinks of staphylococci (16). Thus, this analysis suggests that Tse4 is likely a bifunctional (lytic transglycosylase and endopeptidase) effector capable of targeting the cell wall of various bacteria.
Fig. 5.
Tse4 is a bifunctional T6SS effector with modular architecture. (A) PG composition of bacterial species susceptible to Tse4. (B) Chromatograms of muropeptides released following PG treatment with the indicated enzymes. M4 = Tetra, D44 = TetraTetra, D44N = TetraTetraAnhydro, and M4N = TetraAnhydro. (C) Domain architecture of various Tse4-like T6SS effectors. Effector locus tags are indicated.
To determine whether Tse4 possesses lytic transglycosylase and endopeptidase activities, we performed in vitro enzymatic assays followed by analysis of the solubilized muropeptides (PG fragments) by Ultra Performance Liquid Chromatography and Mass Spectrometry. Muramidase digestion of the PG produces a profile where the main peaks (muropeptides) are the monomeric disaccharide tetrapeptide (M4) and its dimeric (crosslinked) form (D44 and D44N) (Fig. 5B and SI Appendix, Fig. S10). Subsequent addition of Tse4 converts the dimers to monomers (M4 and M4N), thereby confirming its endopeptidase activity. To test the putative lytic transglycosylase activity of Tse4, we incubated the PG first with the endopeptidase ShyA, which breaks the crosslinks to produce PG chains of diverse length and always ending with anhydro-M4 (M4N). Subsequent addition of Tse4 further processes the chains to shorter ones of 1 and 2 disaccharides long (M4N and M4-M4N) (Fig. 5B and SI Appendix, Fig. S11), thereby confirming the lytic transglycosylase activity. As Tse4 cannot further break M4-M4N, we conclude that the predicted Lyz-like domain has endolytic transglycosylase activity. These results were recapitulated by digesting PG with Tse4 alone (Fig. 5B and SI Appendix, Fig. S11). The identity of M4-M4N was confirmed both by MS and by the observation that this chain can be cleaved by muramidase into M4 and M4N (Fig. 5B and SI Appendix, Table S1). Together, these data indicate that Tse4 is an identified bifunctional T6SS effector.

Modularly Organized Bifunctional Effectors Are Present in Various Bacterial Species.

A bioinformatics analysis identified orthologs of Tse4 in various Acinetobacter, Klebsiella, Yersinia, and Enterobacter strains (Fig. 5C). Furthermore, we identified a series of T6SS effectors displaying a modular organization in which the endopeptidase, lytic transglycosylase, and PG-binding domain are linked in different arrangements (Fig. 5C). For example, a putative T6SS effector of Burkholderia spp. contains the same three domains but in the reverse order. Additional related effectors were identified in E. coli and Salmonella enterica. Thus, Tse4 belongs to a broadly distributed class of T6SS effectors that share a similar modular architecture. These findings suggest that Tse4-like effectors are important mediators of antagonistic bacterial interactions. In general, the modular arrangement of these newly discovered T6SS effectors resembles the organization of phage endolysins. Endolysins appear to have acquired multiple activities to enable host diversification and facilitate adaptation to specific growth phase- or strain-specific cell wall modifications (17). It is tempting to speculate that multicatalytic T6SS effectors evolved to provide a fitness advantage against diverse bacterial competitors. We propose that the remarkably broad target range and high potency of Tse4 is due to its dual catalytic activity, which might act synergistically to cause localized, lethal damage in the prey cell wall and/or to adjust to the variable PG chemistries of diverse prey.

Discussion

Early work in V. cholerae and P. aeruginosa suggested that the T6SS targets Gram-negative but not Gram-positive bacteria (18, 19). It was proposed that the PG of Gram-positive bacteria is too thick to allow the T6SS machinery to deliver toxic effectors at effective concentrations. However, recent work demonstrated that the T6SSs of Serratia marcescens and Klebsiella pneumoniae mediate fungal cell death (20, 21), indicating that T6SS machines are able to penetrate and deliver toxic effectors across the fungal cell wall (>110 nm), which is thicker than the cell wall of Gram-positive bacteria (<80 nm). In this work, we show that Ab17978 employs its bifunctional T6SS PG-targeting effector Tse4 to kill various Gram-positive bacteria, including B. subtilis, L. monocytogenes, and S. aureus. In contrast to Gram-negative bacteria, which have an outer membrane shielding their cell wall, Gram-positive bacteria have an exposed PG layer. Between the cell membrane and the PG there is a space, about 30 nm thick, known as the “inner wall zone” (IWZ). TelC, a T7SS effector targeting cell wall synthesis in Gram-positive bacteria, exerts its toxicity in the IWZ of the prey (22). The addition of purified Tse4 to B. subtilis cultures did not induce cell lysis, and coincubation of Ab17978 and B. subtilis in liquid media did not result in bacterial killing, indicating that the T6SS machinery is required for the delivery of Tse4. We speculate that Tse4 is delivered into the IWZ by the T6SS apparatus. Alternatively, Tse4 could be translocated into the cytoplasm and subsequently exported through the cytoplasmic membrane, a mechanism that has been shown for the VgrG3 effector of V. cholerae. Future work will elucidate how Tse4 exerts its toxicity against the Gram-positive prey.
The outcome of interbacterial interactions is impacted by the environmental conditions in which these interactions occur (9). Here, we show that by secreting D-Lys, Ab17978 modifies its microenvironment to potentiate Tse4 activity and increase T6SS-dependent killing of Gram-negative and Gram-positive prey. Our data suggest that the synergistic effect of D-Lys on Tse4 lethality is likely due to alkalization of the predator-prey zone of contact. Consistently, we found that Tse4 has enhanced activity under alkaline conditions. Thus, our work establishes a class of bifunctional, modularly organized T6SS effectors that are effective against Gram-positive and Gram-negative bacteria; highlights the role of D-amino acids in modulating the microenvironment; and redefines the function of the T6SS to include the warfare between Gram-negative and Gram-positive bacteria. Although it is not possible to predict what Gram-positive bacteria Acinetobacter spp. will encounter in the environment, Acinetobacter strains are likely involved in antagonistic interactions with Gram-positive pathogens such as Enterococcus, Staphylococcus, and Streptococcus species, which are known to coexist in polymicrobial infections (2325). Future work will be necessary to better understand the full scope of T6SS-mediated killing of Gram-negative and Gram-positive bacteria and elucidate the effect of these interactions in shaping the composition of bacterial communities in the context of the human microbiota and polymicrobial infections.

Materials and Methods

The bacterial strains and plasmids used in this study are listed in the SI Appendix, Tables S1 and S2. Bacterial killing assays were performed as described in the SI Appendix, Materials and Methods. M-cresol purple (50 µg/mL) and phenol red (18 µg/mL) were used to monitored local pH change due to Ab17978 growth in solid media. Bacterial growth on solid media were quantified by colony-forming unit (CFU) enumeration and Western Blotting using anti-glycan antibody as described in the SI Appendix, Materials and Methods. 6XHistag recombinant Tse4 were purified as described in the SI Appendix, Materials and Methods. PG isolation and Tse4 in vitro reaction were performed as described in the SI Appendix, Materials and Methods. A full description of methods is available in the SI Appendix, Materials and Methods. All data are available in the main text or the SI Appendix, Materials and Methods.

Data Availability

All study data are included in the article and/or SI Appendix.

Acknowledgments

This work was supported by NIH Grant 1R01AI125363-01 and 5R21AI137188-02 to M.F.F. Work in the F.C. laboratory was supported by the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation, the Laboratory of Molecular Infection Medicine Sweden, and the Kempe Foundation. We thank Dr. Ichiro Matsumura (Emory University) for providing B. subtilis SCK6 harboring pBAV1k-T5-gfp plasmid; Dr. Laura Alvarez for providing ShyA; and Dr. Clay Jackson-Litteken for critical reading of the manuscript.

Supporting Information

Appendix (PDF)

References

1
S. J. Hersch, K. Manera, T. G. Dong, Defending against the type six secretion system: Beyond immunity genes the bacterial type six secretion system (T6SS) delivers toxic effector proteins into neighboring cells. Cell Rep. 33, 108259 (2020).
2
N. H. Le et al., Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare. Sci. Adv. 6, eabb5614 (2020).
3
W. P. J. Smith et al., The evolution of the type VI secretion system as a disintegration weapon. PLoS Biol. 18, e3000720 (2020).
4
A. B. Russell et al., Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475, 343–347 (2011).
5
J. C. Whitney et al., Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair. J. Biol. Chem. 288, 26616–26624 (2013).
6
J. Ma, M. Sun, Z. Pan, C. Lu, H. Yao, Diverse toxic effectors are harbored by vgrG islands for interbacterial antagonism in type VI secretion system. Biochim. Biophys. Acta, Gen. Subj. 1862, 1635–1643 (2018).
7
A. B. Russell et al., A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11, 538–549 (2012).
8
A. Aliashkevich, L. Alvarez, F. Cava, New insights into the mechanisms and biological roles of D-amino acids in complex eco-systems. Front. Microbiol. 9, 683 (2018).
9
K. D. LaCourse et al., Conditional toxicity and synergy drive diversity among antibacterial effectors. Nat. Microbiol. 3, 440–446 (2018).
10
B. S. Weber et al., Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis. MBio 7, e01253-16 (2016).
11
F. Cava, M. A. de Pedro, H. Lam, B. M. Davis, M. K. Waldor, Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 30, 3442–3453 (2011).
12
J. C. Wilks, J. L. Slonczewski, pH of the cytoplasm and periplasm of Escherichia coli: Rapid measurement by green fluorescent protein fluorimetry. J. Bacteriol. 189, 5601–5607 (2007).
13
J. C. Whitney et al., Genetically distinct pathways guide effector export through the type VI secretion system. Mol. Microbiol. 92, 529–542 (2014).
14
W. Vollmer, D. Blanot, M. A. de Pedro, Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008).
15
T. Dörr, F. Cava, H. Lam, B. M. Davis, M. K. Waldor, Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae. Mol. Microbiol. 89, 949–962 (2013).
16
M. Firczuk, A. Mucha, M. Bochtler, Crystal structures of active LytM. J. Mol. Biol. 354, 578–590 (2005).
17
H. Oliveira et al., Molecular aspects and comparative genomics of bacteriophage endolysins. J. Virol. 87, 4558–4570 (2013).
18
D. L. MacIntyre, S. T. Miyata, M. Kitaoka, S. Pukatzki, The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc. Natl. Acad. Sci. U.S.A. 107, 19520–19524 (2010).
19
S. Chou et al., Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system. Cell Rep. 1, 656–664 (2012).
20
K. Trunk et al., The type VI secretion system deploys antifungal effectors against microbial competitors. Nat. Microbiol. 3, 920–931 (2018).
21
D. Storey et al., Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent. PLoS Pathog. 16, e1007969 (2020).
22
T. A. Klein, M. Pazos, M. G. Surette, W. Vollmer, J. C. Whitney, Molecular basis for immunity protein recognition of a type VII secretion system exported antibacterial toxin. J. Mol. Biol. 430, 4344–4358 (2018).
23
C. H. Yo et al., Clinical predictors and outcome impact of community-onset polymicrobial bloodstream infection. Int. J. Antimicrob. Agents 54, 716–722 (2019).
24
R. A. Heitkamp et al., Association of Enterococcus spp. with severe combat extremity injury, intensive care, and polymicrobial wound infection. Surg. Infect. (Larchmt.) 19, 95–103 (2018).
25
N. Castellanos et al., A study on Acinetobacter baumannii and Staphylococcus aureus strains recovered from the same infection site of a diabetic patient. Curr. Microbiol. 76, 842–847 (2019).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 118 | No. 40
October 5, 2021
PubMed: 34588306

Classifications

Data Availability

All study data are included in the article and/or SI Appendix.

Submission history

Accepted: August 20, 2021
Published online: September 29, 2021
Published in issue: October 5, 2021

Keywords

  1. T6SS
  2. peptidoglycan
  3. effector
  4. microenvironment

Acknowledgments

This work was supported by NIH Grant 1R01AI125363-01 and 5R21AI137188-02 to M.F.F. Work in the F.C. laboratory was supported by the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation, the Laboratory of Molecular Infection Medicine Sweden, and the Kempe Foundation. We thank Dr. Ichiro Matsumura (Emory University) for providing B. subtilis SCK6 harboring pBAV1k-T5-gfp plasmid; Dr. Laura Alvarez for providing ShyA; and Dr. Clay Jackson-Litteken for critical reading of the manuscript.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110;
Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, 90187 Umeå, Sweden
Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110;
Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, 90187 Umeå, Sweden
Mario F. Feldman1 [email protected]
Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110;

Notes

1
To whom correspondence may be addressed. Email: [email protected].
Author contributions: N.-H.L., V.P., F.C., and M.F. designed research; N.-H.L., V.P., J.L., and F.C. performed research; N.-H.L., V.P., F.C., and M.F. analyzed data; and N.-H.L., J.L., F.C., and M.F. wrote the paper.

Competing Interests

The authors declare no competing interest.

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