Structure and mechanism of biosynthesis of Streptococcus mutans cell wall polysaccharide.

Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c- specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a repeating →3)α-Rha(1→2)α-Rha(1→ polyrhamnose backbone, with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one major and two minor Glc modifications. The major Glc modification, α-Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are ß-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and α-Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor ß-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.

O-glycosylation of intrinsically disordered regions regulates homeostasis of membrane proteins in streptococci.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O-glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae, and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans. Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans. We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O-glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O-glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intra-species diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low inter-species and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Further, we observe subtype-specific effects of GBS T7SS on host colonization, as subtype I but not subtype III T7SS promotes GBS vaginal persistence. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Functional Analysis of EspM, an ESX-1-Associated Transcription Factor in Mycobacterium marinum.

Pathogenic mycobacteria use the ESX-1 secretion system to escape the macrophage phagosome and survive infection. We demonstrated that the ESX-1 system is regulated by feedback control in Mycobacterium marinum, a nontuberculous pathogen and model for the human pathogen Mycobacterium tuberculosis. In the presence of a functional ESX-1 system, the WhiB6 transcription factor upregulates expression of ESX-1 substrate genes. In the absence of an assembled ESX-1 system, the conserved transcription factor, EspM, represses whiB6 expression by specifically binding the whiB6 promoter. Together, WhiB6 and EspM fine-tune the levels of ESX-1 substrates in response to the secretion system. The mechanisms underlying control of the ESX-1 system by EspM are unknown. Here, we conduct a structure and function analysis to investigate how EspM is regulated. Using biochemical approaches, we measured the formation of higher-order oligomers of EspM in vitro. We demonstrate that multimerization in vitro can be mediated through multiple domains of the EspM protein. Using a bacterial monohybrid system, we showed that EspM self-associates through multiple domains in Escherichia coli. Using this system, we performed a genetic screen to identify EspM variants that failed to self-associate. The screen yielded four EspM variants of interest, which we tested for activity in M. marinum. Our study revealed that the two helix-turn-helix domains are functionally distinct. Moreover, the helix bundle domain is required for wild-type multimerization in vitro. Our data support models where EspM monomers or hexamers contribute to the regulation of whiB6 expression. IMPORTANCE Pathogenic mycobacteria are bacteria that pose a large burden to human health globally. The ESX-1 secretion system is required for pathogenic mycobacteria to survive within and interact with the host. Proper function of the ESX-1 secretion system is achieved by tightly controlling the expression of secreted virulence factors, in part through transcriptional regulation. Here, we characterize the conserved transcription factor EspM, which regulates the expression of ESX-1 virulence factors. We define domains required for EspM to form multimers and bind DNA. These findings provide an initial characterization an ESX-1 transcription factor and provide insights into its mechanism of action.

PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

Modification of cell wall polysaccharide guides cell division in Streptococcus mutans.

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.

Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

PE5-PPE4-EspG3 heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems.

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between "open" and "closed" forms, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE-PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE-PPE pairs.

Type VII Secretion Substrates of Pathogenic Mycobacteria Are Processed by a Surface Protease.

Tuberculosis, one of the world's most severe infectious diseases, is caused by Mycobacterium tuberculosis A major weapon of this pathogen is a unique cell wall that protects the pathogen from eradication by the immune system. Mycobacteria have specialized secretion systems, e.g., type VII secretion or ESX systems, to transport substrates across this cell wall. The largest group of proteins that are secreted by these ESX systems are the PE proteins. Previously, it was shown that the N-terminal PE domain of about 100 amino acids is required for secretion. Here, we describe the identification of an aspartic protease, designated PecA, that removes (part of) this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.IMPORTANCE Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria (N. D. Rawlings and A. Bateman, BMC Genomics 10:437, 2009, https://doi.org/10.1186/1471-2164-10-437). In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis In addition, pathogenicity of M. marinum is reduced in the ΔpecA mutant, indicating that PecA contributes to virulence.

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides.

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.

Novel Antimycobacterial Compounds Suppress NAD Biogenesis by Targeting a Unique Pocket of NaMN Adenylyltransferase.

Conventional treatments to combat the tuberculosis (TB) epidemic are falling short, thus encouraging the search for novel antitubercular drugs acting on unexplored molecular targets. Several whole-cell phenotypic screenings have delivered bioactive compounds with potent antitubercular activity. However, their cellular target and mechanism of action remain largely unknown. Further evaluation of these compounds may include their screening in search for known antitubercular drug targets hits. Here, a collection of nearly 1400 mycobactericidal compounds was screened against Mycobacterium tuberculosis NaMN adenylyltransferase ( MtNadD), a key enzyme in the biogenesis of NAD cofactor that was recently validated as a new drug target for dormant and active tuberculosis. We found three chemotypes that efficiently inhibit MtNadD in the low micromolar range in vitro. SAR and cheminformatics studies of commercially available analogues point to a series of benzimidazolium derivatives, here named N2, with bactericidal activity on different mycobacteria, including M. abscessus, multidrug-resistant M. tuberculosis, and dormant M. smegmatis. The on-target activity was supported by the increased resistance of an M. smegmatis strain overexpressing the target and by a rapid decline in NAD(H) levels. A cocrystal structure of MtNadD with N2-8 inhibitor reveals that the binding of the inhibitor induced the formation of a new quaternary structure, a dimer-of-dimers where two copies of the inhibitor occupy symmetrical positions in the dimer interface, thus paving the way for the development of a new generation of selective MtNadD bioactive inhibitors. All these results strongly suggest that pharmacological inhibition of MtNadD is an effective strategy to combat dormant and resistant Mtb strains.

Architecture, Function, and Substrates of the Type II Secretion System.

The type II secretion system (T2SS) delivers toxins and a range of hydrolytic enzymes, including proteases, lipases, and carbohydrate-active enzymes, to the cell surface or extracellular space of Gram-negative bacteria. Its contribution to survival of both extracellular and intracellular pathogens as well as environmental species of proteobacteria is evident. This dynamic, multicomponent machinery spans the entire cell envelope and consists of a cytoplasmic ATPase, several inner membrane proteins, a periplasmic pseudopilus, and a secretin pore embedded in the outer membrane. Despite the trans-envelope configuration of the T2S nanomachine, proteins to be secreted engage with the system first once they enter the periplasmic compartment via the Sec or TAT export system. Thus, the T2SS is specifically dedicated to their outer membrane translocation. The many sequence and structural similarities between the T2SS and type IV pili suggest a common origin and argue for a pilus-mediated mechanism of secretion. This minireview describes the structures, functions, and interactions of the individual T2SS components and the general architecture of the assembled T2SS machinery and briefly summarizes the transport and function of a growing list of T2SS exoproteins. Recent advances in cryo-electron microscopy, which have led to an increased understanding of the structure-function relationship of the secretin channel and the pseudopilus, are emphasized.

The Mycobacterium tuberculosis Pup-proteasome system regulates nitrate metabolism through an essential protein quality control pathway.

The human pathogen Mycobacterium tuberculosis encodes a proteasome that carries out regulated degradation of bacterial proteins. It has been proposed that the proteasome contributes to nitrogen metabolism in M. tuberculosis, although this hypothesis had not been tested. Upon assessing M. tuberculosis growth in several nitrogen sources, we found that a mutant strain lacking the Mycobacterium proteasomal activator Mpa was unable to use nitrate as a sole nitrogen source due to a specific failure in the pathway of nitrate reduction to ammonium. We found that the robust activity of the nitrite reductase complex NirBD depended on expression of the groEL/groES chaperonin genes, which are regulated by the repressor HrcA. We identified HrcA as a likely proteasome substrate, and propose that the degradation of HrcA is required for the full expression of chaperonin genes. Furthermore, our data suggest that degradation of HrcA, along with numerous other proteasome substrates, is enhanced during growth in nitrate to facilitate the derepression of the chaperonin genes. Importantly, growth in nitrate is an example of a specific condition that reduces the steady-state levels of numerous proteasome substrates in M. tuberculosis.

Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems.

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central ß-sheet and two α-helical bundles. In addition, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3, and EspG5 chaperones using small-angle X-ray scattering reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the small-angle X-ray scattering analysis.

Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site.

Mycobacterium tuberculosis is the cause of the world's most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetX converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetX proteins, including M. tuberculosis (MtMetX), Mycolicibacterium abscessus (MaMetX), and Mycolicibacterium hassiacum (MhMetX). A comparison of homoserine transacetylases from other bacterial and fungal species reveals a high degree of structural conservation amongst the enzymes. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetX. The deep active-site tunnel surrounding the catalytic serine yielded many consensus clusters during mapping, suggesting that MtMetX is highly druggable.

Targeting phosphatases of regenerating liver (PRLs) in cancer.

The phosphatase of regenerating liver (PRL) family, also known as protein tyrosine phosphatase 4A (PTP4A), are dual-specificity phosphatases with largely unknown cellular functions. However, accumulating evidence indicates that PRLs are oncogenic across a broad variety of human cancers. PRLs are highly expressed in advanced tumors and metastases compared to early stage cancers or matched healthy tissue, and high expression of PRLs often correlates with poor patient prognosis. Consequentially, PRLs have been considered potential therapeutic targets in cancer. Persistent efforts have been made to define their role and mechanism in cancer progression and to create specific PRL inhibitors for basic research and drug development. However, targeting PRLs with small molecules remains challenging due to the highly conserved active site of protein tyrosine phosphatases and a high degree of sequence similarity between the PRL protein families. Here, we review the current PRL inhibitors, including the strategies used for their identification, their biological efficacy, potency, and selectivity, with a special focus on how PRL structure can inform future efforts to develop specific PRL inhibitors.

Structural and functional insights into the role of BamD and BamE within the ß-barrel assembly machinery in Neisseria gonorrhoeae.

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes.

In many Lactobacillales species (i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes, synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N-acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall.