Drivers of geophagy by red brocket deer (Mazama americana) at Amazonian interior forest mineral licks.

Mineral licks are key ecological components of the Amazon rainforest, providing critical dietary functions for herbivorous and frugivorous mammals and birds, which help maintain the structure and function of the forest itself through seed and nutrient dispersal. One of the most frequent visitors of interior forest mineral licks in the Amazon is the red brocket deer (Mazama americana), a large-bodied ruminant frugivore and seed predator. While several hypotheses for the drivers of geophagy exist, including mineral supplementation, toxin adsorption, and habitat selection, robust data on geophagy for the red brocket deer for large numbers of mineral licks is nonexistent. We used soil data from 83 mineral licks in conjunction with camera trap data from 52 of those mineral licks and a mixed-effects modeling approach to test the three proposed hypotheses of geophagy for the red brocket deer. We found that consumed soils at mineral licks had elevated concentrations of almost all major and minor biologically active minerals measured, including Ca, Na, Mg, K, Cu, Zn, and Mn. Model results suggest that all three hypotheses hold true to some extent for the red brocket deer, with the greatest support for the mineral supplementation hypothesis, in particular with respect to Mg, Ca, Na, Cu, and Zn. This study provides critical information on the feeding ecology of the red brocket deer in the wild, and the first robust analysis of geophagy of an Amazonian mammal involving a large sample size of interior forest mineral licks.

Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Escherichia coli CadB is capable of promiscuously transporting muropeptides and contributing to peptidoglycan recycling.

The bacterial peptidoglycan (PG) cell wall is remodeled during growth and division, releasing fragments called muropeptides. Muropeptides can be internalized and reused in a process called PG recycling. Escherichia coli is highly devoted to recycling muropeptides and is known to have at least two transporters, AmpG and OppBCDF, that import them into the cytoplasm. While studying mutants lacking AmpG, we unintentionally isolated mutations that led to the altered expression of a third transporter, CadB. CadB is normally upregulated under acidic pH conditions and is an antiporter for lysine and cadaverine. Here, we explored if CadB was altering PG recycling to assist in the absence of AmpG. Surprisingly, CadB overexpression was able to restore PG recycling when both AmpG and OppBCDF were absent. CadB was found to import freed PG peptides, a subpopulation of muropeptides, through a promiscuous activity. Altogether, our data support that CadB is a third transporter capable of contributing to PG recycling. IMPORTANCE Bacteria produce a rigid mesh cell wall. During growth, the cell wall is remodeled, which releases cell wall fragments. If released into the extracellular environment, cell wall fragments can trigger inflammation by the immune system of a host. Gastrointestinal bacteria, like Escherichia coli, have dedicated pathways to recycle almost all cell wall fragments they produce. E. coli contains two known recycling transporters, AmpG and Opp, that we previously showed are optimized for growth in different environments. Here, we identify that a third transporter, CadB, can also contribute to cell wall recycling. This work expands our understanding of cell wall recycling and highlights the dedication of organisms like E. coli to ensure high recycling in multiple growth environments.

Characterization of the resistome and predominant genetic lineages of Gram-positive bacteria causing keratitis.

Bacterial keratitis is a vision-threatening infection mainly caused by Gram-positive bacteria (GPB). Antimicrobial therapy is commonly empirical using broad-spectrum agents with efficacy increasingly compromised by the emergence of antimicrobial resistance. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of GPB causing keratitis and to characterize their antimicrobial resistance patterns. A total of 161 isolates, including Staphylococcus aureus (n = 86), coagulase-negative staphylococci (CoNS; n = 34), Streptococcus spp. (n = 34), and Enterococcus faecalis (n = 7), were included. The population of S. aureus isolates consisted mainly of clonal complex 5 (CC5) (30.2%). Similarly, the population of Staphylococcus epidermidis was homogenous with most of them belonging to CC2 (78.3%). Conversely, the genetic population of Streptococcus pneumoniae was highly diverse. Resistance to first-line antibiotics was common among staphylococci, especially among CC5 S. aureus. Methicillin-resistant S. aureus was commonly resistant to fluoroquinolones and azithromycin (78.6%) and tobramycin (57%). One-third of the CoNS were resistant to fluoroquinolones and 53% to azithromycin. Macrolide resistance was commonly caused by erm genes in S. aureus, mphC and msrA in CoNS, and mefA and msr(D) in streptococci. Aminoglycoside resistance in staphylococci was mainly associated with genes commonly found in mobile genetic elements and that encode for nucleotidyltransferases like ant(4')-Ib and ant(9)-Ia. Fluroquinolone-resistant staphylococci carried from 1 to 4 quinolone resistance-determining region mutations, mainly in the gyrA and parC genes. We found that GPB causing keratitis are associated with strains commonly resistant to first-line topical therapies, especially staphylococcal isolates that are frequently multidrug-resistant and associated with major hospital-adapted epidemic lineages.

A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

Staphylococcus aureus activates NRLP3-dependent IL-1ß secretion from human conjunctival goblet cells using α toxin and toll-like receptors 2 and 1.

We used cultured human conjunctival goblet cells to determine (i) whether the toxigenic S. aureus- induced activation of the epithelial goblet cells requires two signals to activate the NLRP3 inflammasome, (ii) if one signal is mediated by TLR1, TLR2, or TLR6, and (iii) if the S. aureus toxin α toxin is another signal for the activation of the inflammasome and secretion of mature IL-1ß. Cultured cells were incubated with siRNA to knock down the different TLRs. After stimulation with toxigenic S. aureus RN6390, pro-IL-1ß synthesis, caspase-1 activity, and mature IL-1ß secretion were measured. In a separate set of experiments, the cells were incubated with toxigenic S. aureus RN6390 or mutant S. aureus ALC837 that does not express α toxin with or without exogenous α toxin. A gentamicin protection assay was used to determine if intracellular bacteria were active. We conclude that α toxin from toxigenic S. aureus triggers two separate mechanisms required for the activation of the NLRP3 inflammasome and secretion of mature IL-1ß. In the first mechanism, α toxin secreted from internalized S. aureus produces a pore, allowing the internalized bacteria and associated pathogen-associated molecular patterns to interact with intracellular TLR2 and, to a lesser extent, TLR1. In the second mechanism, α toxin forms a pore in the plasma membrane, leading to an efflux of cytosolic K+ and influx of Ca2+. We conclude that α toxin by these two different mechanisms triggers the synthesis of pro-IL-1ß and NLRP3 components, activation of capase-1, and secretion of mature IL-1ß to defend against bacterial infection.

Bacterial cell surface nanoenvironment requires a specialized chaperone to activate a peptidoglycan biosynthetic enzyme.

Bacillus subtilis spores are produced inside the cytosol of a mother cell. Spore surface assembly requires the SpoVK protein in the mother cell, but its function is unknown. Here, we report that SpoVK is a dedicated chaperone from a distinct higher-order clade of AAA+ ATPases that activates the peptidoglycan glycosyltransferase MurG during sporulation, even though MurG does not normally require activation by a chaperone during vegetative growth. MurG redeploys to the spore surface during sporulation, where we show that the local pH is reduced and propose that this change in cytosolic nanoenvironment necessitates a specific chaperone for proper MurG function. Further, we show that SpoVK participates in a developmental checkpoint in which improper spore surface assembly inactivates SpoVK, which leads to sporulation arrest. The AAA+ ATPase clade containing SpoVK includes other dedicated chaperones involved in secretion, cell-envelope biosynthesis, and carbohydrate metabolism, suggesting that such fine-tuning might be a widespread feature of different subcellular nanoenvironments.

Escherichia coli utilizes multiple peptidoglycan recycling permeases with distinct strategies of recycling.

Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the existing layer of PG is remodeled and PG fragments are released. Enterics such as Escherichia coli go to great lengths to internalize and reutilize PG fragments. E. coli is estimated to break down one-third of its cell wall, yet only loses ~0 to 5% of meso-diaminopimelic acid, a PG-specific amino acid, per generation. Two transporters were identified early on to possibly be the primary permease that facilitates PG fragment recycling, i) AmpG and ii) the Opp ATP binding cassette transporter in conjunction with a PG-specific periplasmic binding protein, MppA. The contribution of each transporter to PG recycling has been debated. Here, we have found that AmpG and MppA/Opp are differentially regulated by carbon source and growth phase. In addition, MppA/Opp is uniquely capable of high-affinity scavenging of muropeptides from growth media, demonstrating that AmpG and MppA/Opp allow for different strategies of recycling PG fragments. Altogether, this work clarifies environmental contexts under which E. coli utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.

Gut microbial fatty acid isomerization modulates intraepithelial T cells.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4+ intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4+CD8αα+ IELs in gnotobiotic mice. Restoration of CLAs increases CD4+CD8αα+ IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4+CD8αα+ IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4+ T cells that were CD4+CD8αα+.

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters.

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous or homologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of protein-encoding ortholog groups for individual genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of statistics for each inferred ortholog group. These programs are showcased through application to: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-genetic insights of two common BGCs in a fungal species, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

Microbiology of Eye Infections at the Massachusetts Eye and Ear: An 8-Year Retrospective Review Combined With Genomic Epidemiology.

PURPOSE: Ocular bacterial infections are important causes of morbidity and vision loss. Early antimicrobial therapy is necessary to save vision, but their efficacy is increasingly compromised by antimicrobial resistance (AMR). We assessed the etiology of ocular bacterial infections seen at Massachusetts Eye and Ear and investigated the molecular epidemiology and AMR profiles of contemporary isolates. DESIGN: Laboratory investigation. METHODS: We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of leading ocular pathogens and their AMR profiles. RESULTS: A total of 1601 isolates were collected from 2014 to 2021, with Staphylococcus aureus (n = 621), coagulase-negative staphylococci (CoNS) (n = 234), Pseudomonas aeruginosa (n = 213), Enterobacteriaceae (n = 167), and Streptococcus pneumoniae (n = 95) being the most common. Resistance was high among staphylococci, with methicillin resistance (MR) detected in 28% of S aureus and 39.8% of CoNS isolates. Multidrug resistance (MDR) was frequent among MR staphylococci (MRSA 60%, MRCoNS 76.1%). The population of S aureus isolates consisted mainly of 2 clonal complexes (CCs): CC8 (26.1%) and CC5 (24.1%). CC5 strains carried a variety of AMR markers, resulting in high levels of resistance to first-line therapies. Similarly, the population of ocular Staphylococcus epidermidis was homogenous with most belonging to CC2 (85%), which were commonly MDR (48%). Conversely, ocular S pneumoniae, P aeruginosa, and Enterobacteriaceae were often susceptible to first-line therapies and grouped into highly diverse genetic populations. CONCLUSION: Our data showed that ocular bacterial infections in our patient population are disproportionately caused by strains that are resistant to clinically relevant antibiotics and are associated with major epidemic genotypes with both community and hospital associations.

Global diversity of enterococci and description of 18 novel species.

Enterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species, Enterococcus faecalis and E. faecium uniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool of Enterococcus-adapted genes from which known facile gene exchangers such as E. faecalis and E. faecium may draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition. E. faecalis and E. faecium were isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus, potential threats to human health, and new insights into its evolution.

Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila.

Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular Pathogens.

PURPOSE: Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection. DESIGN: Experimental laboratory investigation. METHODS: A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in a pilot clinical study testing samples from infectious retinitis (n = 15) and endophthalmitis (n = 12) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture. RESULTS: Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens. CONCLUSION: This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular fluids that could significantly improve diagnostics of sight-threatening intraocular infections.

Physical, landscape, and chemical properties of Amazonian interior forest mineral licks.

Mineral licks, sites where animals go to consume soil, are key resources for herbivorous birds and mammals in the Amazon, providing supplemental dietary nutrients and toxin adsorption functions. However, because they are often difficult to find, the properties of mineral licks are poorly understood. Here, we undertake the largest survey of Amazonian mineral licks to date to determine the landscape, physical, and chemical properties of these critical sites. We used a generalized linear mixed-effects modeling framework to assess how soil samples from 83 mineral licks differ from nearby control soils in a series of physical and chemical characteristics, then used Jaccard's index of similarity and a principal component analysis (PCA) to determine how those samples differed among themselves. We found that mineral licks were generally located in specific ranges of landscape variables. Soils from mineral licks had elevated concentrations of almost all minerals measured. There was very little similarity between consumed and control samples, and within each sample type. We suggest that these mineral licks have the potential to provide multiple services to visiting species, demonstrating their ecological importance. The high levels of dissimilarity between samples indicate that a large sample of mineral licks is needed to draw conclusions in studies pertaining to geophagy. We emphasize that studying mammal and bird visitation at these sites could provide critical conservation and physiological information on cryptic and understudied species of Amazonian herbivores.

Peptidoglycan recycling mediated by an ABC transporter in the plant pathogen Agrobacterium tumefaciens.

During growth and division, the bacterial cell wall peptidoglycan (PG) is remodelled, resulting in the liberation of PG muropeptides which are typically reinternalized and recycled. Bacteria belonging to the Rhizobiales and Rhodobacterales orders of the Alphaproteobacteria lack the muropeptide transporter AmpG, despite having other key PG recycling enzymes. Here, we show that an alternative transporter, YejBEF-YepA, takes over this role in the Rhizobiales phytopathogen Agrobacterium tumefaciens. Muropeptide import by YejBEF-YepA governs expression of the ß-lactamase AmpC in A. tumefaciens, contributing to ß-lactam resistance. However, we show that the absence of YejBEF-YepA causes severe cell wall defects that go far beyond lowered AmpC activity. Thus, contrary to previously established Gram-negative models, PG recycling is vital for cell wall integrity in A. tumefaciens. YepA is widespread in the Rhizobiales and Rhodobacterales, suggesting that YejBEF-YepA-mediated PG recycling could represent an important but overlooked aspect of cell wall biology in these bacteria.

Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation.

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Population structure of ocular Streptococcus pneumoniae is highly diverse and formed by lineages that escape current vaccines.

Streptococcus pneumoniae is a leading cause of ocular infections including serious and sight-threatening conditions. The use of pneumococcal conjugate vaccines (PCV) has substantially reduced the incidence of pneumonia and invasive pneumococcal diseases, but has had limited impact on ocular infections. Additionally, widespread vaccine use has resulted in ongoing selective pressure and serotype replacement in carriage and disease. To gain insight into the population structure of pneumococcal isolates causing ocular infections in a post-PCV-13 time period, we investigated the genomic epidemiology of ocular S. pneumoniae isolates (n=45) collected at Massachusetts Eye and Ear between 2014 and 2017. By performing a series of molecular typing methods from draft genomes, we found that the population structure of ocular S. pneumoniae is highly diverse with 27 sequence types (grouped into 18 clonal complexes) and 17 serotypes being identified. Distribution of these lineages diverged according to the site of isolation, with conjunctivitis being commonly caused by isolates grouped in the Epidemic Conjunctivitis Cluster-ECC (60 %), and ST448 (53.3 %) being most frequently identified. Conversely, S. pneumoniae keratitis cases were caused by a highly diverse population of isolates grouping within 15 different clonal complexes. Serotyping inference demonstrated that 95.5 % of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80 %) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2 %) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2 %), and usually associated with the mefA +msrD genotype (n=15). These genes were located in the Macrolide Efflux Genetic Assembly cassette and were associated with low-level in vitro resistance to 14- and 15-membered macrolides. Less frequently, macrolide-resistant isolates carried an ermB gene (n=4), which was co-located with the tetM gene in a Tn-916-like transposon. Our study demonstrates that the population structure of ocular S. pneumoniae is highly diverse, mainly composed by isolates that escape the PCV-13 vaccine, with patterns of tissue/niche segregation, adaptation and specialization. These findings suggest that the population structure of ocular pneumococcus may be shaped by multiple factors including PCV-13 selective pressure, microbial-related and niche-specific host-associated features.

Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Bacterial developmental checkpoint that directly monitors cell surface morphogenesis.

Bacillus subtilis spores are encased in two concentric shells: an outer proteinaceous "coat" and an inner peptidoglycan "cortex," separated by a membrane. Cortex assembly depends on coat assembly initiation, but how cells achieve this coordination across the membrane is unclear. Here, we report that the protein SpoVID monitors the polymerization state of the coat basement layer via an extension to a functional intracellular LysM domain that arrests sporulation when coat assembly is initiated improperly. Whereas extracellular LysM domains bind mature peptidoglycan, SpoVID LysM binds to the membrane-bound lipid II peptidoglycan precursor. We propose that improper coat assembly exposes the SpoVID LysM domain, which then sequesters lipid II and prevents cortex assembly. SpoVID defines a widespread group of firmicute proteins with a characteristic N-terminal domain and C-terminal peptidoglycan-binding domains that might combine coat and cortex assembly roles to mediate a developmental checkpoint linking the morphogenesis of two spatially separated supramolecular structures.