Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae.

Streptococcus pneumoniae, a common colonizer of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy participants of controlled human infection studies. We hypothesized that surface expression of pneumococcal lipoproteins, recognized by the innate immune receptor TLR2, mediates epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signaling. Loss of lgt also led to the concomitant decrease in interferon signaling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in ~10% of global pneumococcal isolates. While palA was enriched in the carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonization is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonized by pneumococci without becoming unwell. It is therefore important to better understand how the colonization process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonization and disease.

Clonal Expansion of a Streptococcus pneumoniae Serotype 3 Capsule Variant Sequence Type 700 With Enhanced Vaccine Escape Potential After 13-Valent Pneumococcal Conjugate Vaccine Introduction.

BACKGROUND: Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage. METHODS: The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015-2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children. RESULTS: Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing. CONCLUSIONS: A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines.

The metabolic, virulence and antimicrobial resistance profiles of colonising Streptococcus pneumoniae shift after PCV13 introduction in urban Malawi.

Streptococcus pneumoniae causes substantial mortality among children under 5-years-old worldwide. Polysaccharide conjugate vaccines (PCVs) are highly effective at reducing vaccine serotype disease, but emergence of non-vaccine serotypes and persistent nasopharyngeal carriage threaten this success. We investigated the hypothesis that following vaccine, adapted pneumococcal genotypes emerge with the potential for vaccine escape. We genome sequenced 2804 penumococcal isolates, collected 4-8 years after introduction of PCV13 in Blantyre, Malawi. We developed a pipeline to cluster the pneumococcal population based on metabolic core genes into "Metabolic genotypes" (MTs). We show that S. pneumoniae population genetics are characterised by emergence of MTs with distinct virulence and antimicrobial resistance (AMR) profiles. Preliminary in vitro and murine experiments revealed that representative isolates from emerging MTs differed in growth, haemolytic, epithelial infection, and murine colonisation characteristics. Our results suggest that in the context of PCV13 introduction, pneumococcal population dynamics had shifted, a phenomenon that could further undermine vaccine control and promote spread of AMR.

Novel Multilocus Sequence Typing and Global Sequence Clustering Schemes for Characterizing the Population Diversity of Streptococcus mitis.

Streptococcus mitis is a common oral commensal and an opportunistic pathogen that causes bacteremia and infective endocarditis; however, the species has received little attention compared to other pathogenic streptococcal species. Effective and easy-to-use molecular typing tools are essential for understanding bacterial population diversity and biology, but schemes specific for S. mitis are not currently available. We therefore developed a multilocus sequence typing (MLST) scheme and defined sequence clusters or lineages of S. mitis using a comprehensive global data set of 322 genomes (148 publicly available and 174 newly sequenced). We used internal 450-bp sequence fragments of seven housekeeping genes (accA, gki, hom, oppC, patB, rlmN, and tsf) to define the MLST scheme and derived the global S. mitis sequence clusters using the PopPUNK clustering algorithm. We identified an initial set of 259 sequence types (STs) and 258 global sequence clusters. The schemes showed high concordance (100%), capturing extensive S. mitis diversity with strains assigned to multiple unique STs and global sequence clusters. The tools also identified extensive within- and between-host S. mitis genetic diversity among isolates sampled from a cohort of healthy individuals, together with potential transmission events, supported by both phylogeny and pairwise single nucleotide polymorphism (SNP) distances. Our novel molecular typing and strain clustering schemes for S. mitis allow for the integration of new strain data, are electronically portable at the PubMLST database (https://pubmlst.org/smitis), and offer a standardized approach to understanding the population structure of S. mitis. These robust tools will enable new insights into the epidemiology of S. mitis colonization, disease and transmission.

The AmiC/NlpD Pathway Dominates Peptidoglycan Breakdown in Neisseria meningitidis and Affects Cell Separation, NOD1 Agonist Production, and Infection.

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In the present study, we demonstrated that N. meningitidis released more PG-derived peptides than PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. The generation of such peptides required the presence of the periplasmic N-acetylmuramyl-l-alanine amidase AmiC and the outer membrane lipoprotein NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly tagged AmiC was found at the septum in some diplococci but was distributed around the cell in most cases. In a human whole-blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis, the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and survival during infection of human blood.

Acute bacterial meningitis.

PURPOSE OF REVIEW: Community-acquired bacterial meningitis is a continually changing disease. This review summarises both dynamic epidemiology and emerging data on pathogenesis. Updated clinical guidelines are discussed, new agents undergoing clinical trials intended to reduce secondary brain damage are presented. RECENT FINDINGS: Conjugate vaccines are effective against serotype/serogroup-specific meningitis but vaccine escape variants are rising in prevalence. Meningitis occurs when bacteria evade mucosal and circulating immune responses and invade the brain: directly, or across the blood-brain barrier. Tissue damage is caused when host genetic susceptibility is exploited by bacterial virulence. The classical clinical triad of fever, neck stiffness and headache has poor diagnostic sensitivity, all guidelines reflect the necessity for a low index of suspicion and early Lumbar puncture. Unnecessary cranial imaging causes diagnostic delays. cerebrospinal fluid (CSF) culture and PCR are diagnostic, direct next-generation sequencing of CSF may revolutionise diagnostics. Administration of early antibiotics is essential to improve survival. Dexamethasone partially mitigates central nervous system inflammation in high-income settings. New agents in clinical trials include C5 inhibitors and daptomycin, data are expected in 2025. SUMMARY: Clinicians must remain vigilant for bacterial meningitis. Constantly changing epidemiology and emerging pathogenesis data are increasing the understanding of meningitis. Prospects for better treatments are forthcoming.

Streptococcal Serine-Rich Repeat Proteins in Colonization and Disease.

Glycosylation of proteins, previously thought to be absent in prokaryotes, is increasingly recognized as important for both bacterial colonization and pathogenesis. For mucosal pathobionts, glycoproteins that function as cell wall-associated adhesins facilitate interactions with mucosal surfaces, permitting persistent adherence, invasion of deeper tissues and transition to disease. This is exemplified by Streptococcus pneumoniae and Streptococcus agalactiae, which can switch from being relatively harmless members of the mucosal tract microbiota to bona fide pathogens that cause life-threatening diseases. As part of their armamentarium of virulence factors, streptococci encode a family of large, glycosylated serine-rich repeat proteins (SRRPs) that facilitate binding to various tissue types and extracellular matrix proteins. This minireview focuses on the roles of S. pneumoniae and S. agalactiae SRRPs in persistent colonization and the transition to disease. The potential of utilizing SRRPs as vaccine targets will also be discussed.

Pan-GWAS of Streptococcus agalactiae Highlights Lineage-Specific Genes Associated with Virulence and Niche Adaptation.

Streptococcus agalactiae (group B streptococcus; GBS) is a colonizer of the gastrointestinal and urogenital tracts, and an opportunistic pathogen of infants and adults. The worldwide population of GBS is characterized by clonal complexes (CCs) with different invasive potentials. CC17, for example, is a hypervirulent lineage commonly associated with neonatal sepsis and meningitis, while CC1 is less invasive in neonates and more commonly causes invasive disease in adults with comorbidities. The genetic basis of GBS virulence and the extent to which different CCs have adapted to different host environments remain uncertain. We have therefore applied a pan-genome-wide association study (GWAS) approach to 1,988 GBS strains isolated from different hosts and countries. Our analysis identified 279 CC-specific genes associated with virulence, disease, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of particular CCs. In CC17 and CC23, for example, we have identified genes encoding pilus, quorum-sensing proteins, and proteins for the uptake of ions and micronutrients which are absent in less invasive lineages. Moreover, in CC17, carriage and disease strains were distinguished by the allelic variants of 21 of these CC-specific genes. Together our data highlight the lineage-specific basis of GBS niche adaptation and virulence.IMPORTANCE GBS is a leading cause of mortality in newborn babies in high- and low-income countries worldwide. Different strains of GBS are characterized by different degrees of virulence, where some are harmlessly carried by humans or animals and others are much more likely to cause disease.The genome sequences of almost 2,000 GBS samples isolated from both animals and humans in high- and low- income countries were analyzed using a pan-genome-wide association study approach. This allowed us to identify 279 genes which are associated with different lineages of GBS, characterized by a different virulence and preferred host. Additionally, we propose that the GBS now carried in humans may have first evolved in animals before expanding clonally once adapted to the human host.These findings are essential to help understand what is causing GBS disease and how the bacteria have evolved and are transmitted.

Attention Seeker: Production, Modification, and Release of Inflammatory Peptidoglycan Fragments in Neisseria Species.

Maintenance of the structural macromolecule peptidoglycan (PG), which involves regulated cycles of PG synthesis and PG degradation, is pivotal for cellular integrity and survival. PG fragments generated from the degradation process are usually efficiently recycled by Gram-negative bacteria. However, Neisseria gonorrhoeae and a limited number of Gram-negative bacteria release PG fragments in amounts sufficient to induce host tissue inflammation and damage during an infection. Due to limited redundancy in PG-modifying machineries and genetic tractability, N. gonorrhoeae serves as a great model organism for the study of biological processes related to PG. This review summarizes the generation, modification, and release of inflammatory PG molecules by N. gonorrhoeae and related species and discusses these findings in the context of understanding bacterial physiology and pathogenesis.

Neisseria gonorrhoeae Crippled Its Peptidoglycan Fragment Permease To Facilitate Toxic Peptidoglycan Monomer Release.

Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency. IMPORTANCE: Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease.

Defects in the flagellar motor increase synthesis of poly-γ-glutamate in Bacillus subtilis.

Bacillus subtilis swims in liquid media and swarms over solid surfaces, and it encodes two sets of flagellar stator homologs. Here, we show that B. subtilis requires only the MotA/MotB stator during swarming motility and that the residues required for stator force generation are highly conserved from the Proteobacteria to the Firmicutes. We further find that mutants that abolish stator function also result in an overproduction of the extracellular polymer poly-γ-glutamate (PGA) to confer a mucoid colony phenotype. PGA overproduction appeared to be the result of an increase in the expression of the pgs operon that encodes genes for PGA synthesis. Transposon mutagenesis was conducted to identify insertions that abolished colony mucoidy and disruptions in known transcriptional regulators of PGA synthesis (Com and Deg two-component systems) as well as mutants defective in transcription-coupled DNA repair (Mfd)-reduced expression of the pgs operon. A final class of insertions disrupted proteins involved in the assembly of the flagellar filament (FliD, FliT, and FlgL), and these mutants did not reduce expression of the pgs operon, suggesting a second mechanism of PGA control.

Peptidoglycan fragment release from Neisseria meningitidis.

Neisseria meningitidis (meningococcus) is a symbiont of the human nasopharynx. On occasion, meningococci disseminate from the nasopharynx to cause invasive disease. Previous work showed that purified meningococcal peptidoglycan (PG) stimulates human Nod1, which leads to activation of NF-κB and production of inflammatory cytokines. No studies have determined if meningococci release PG or activate Nod1 during infection. The closely related pathogen Neisseria gonorrhoeae releases PG fragments during normal growth. These fragments induce inflammatory cytokine production and ciliated cell death in human fallopian tubes. We determined that meningococci also release PG fragments during growth, including fragments known to induce inflammation. We found that N. meningitidis recycles PG fragments via the selective permease AmpG and that meningococcal PG recycling is more efficient than gonococcal PG recycling. Comparison of PG fragment release from N. meningitidis and N. gonorrhoeae showed that meningococci release less of the proinflammatory PG monomers than gonococci and degrade PG to smaller fragments. The decreased release of PG monomers by N. meningitidis relative to N. gonorrhoeae is partly due to ampG, since replacement of gonococcal ampG with the meningococcal allele reduced PG monomer release. Released PG fragments in meningococcal supernatants induced significantly less Nod1-dependent NF-κB activity than released fragments in gonococcal supernatants and tended to induce less interleukin-8 (IL-8) secretion in primary human fallopian tube explants. These results support a model in which efficient PG recycling and extensive degradation of PG fragments lessen inflammatory responses and may be advantageous for maintaining meningococcal carriage in the nasopharynx.