In Vitro Activity of Ertapenem against Neisseria gonorrhoeae Clinical Isolates with Decreased Susceptibility or Resistance to Extended-Spectrum Cephalosporins in Nanjing, China (2013 to 2019).

Neisseria gonorrhoeae isolates collected in Nanjing, China, that possessed decreased susceptibility (or resistance) to extended-spectrum cephalosporins (ESCs) were examined for susceptibility to ertapenem, and their sequence types were determined. Ceftriaxone and cefixime MICs of ≥0.125 mg/L and ≥0.25 mg/L, respectively, were first determined in 259 strains isolated between 2013 and 2019, and then MICs of ertapenem were measured using the antimicrobial gradient Epsilometer test (Etest). Also, genetic determinants of ESC resistance were identified and N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed to analyze associations with ertapenem susceptibility. All isolates displayed ertapenem MICs between 0.006 mg/L and 0.38 mg/L; the overall MIC50 and MIC90 were 0.032 mg/L and 0.125 mg/L, respectively. Forty-four (17.0%) isolates displayed ertapenem MICs of ≥0.125 mg/L; 10 (3.9%) had MICs of ≥0.25 mg/L. The proportion of isolates with ertapenem MICs of ≥0.125 mg/L increased from 4.0% in 2013 to 20.0% in 2019 (χ2 = 24.144, P < 0.001; chi-square test for linear trend). The penA mosaic allele was present in a significantly higher proportion of isolates with ertapenem MICs of ≥0.125 mg/L than of isolates with MICs of ≤0.094 mg/L) (97.7% versus 34.9%, respectively; χ2 = 58.158, P < 0.001). ST5308 was the most prevalent NG-MAST type (8.5%); ST5308 was also significantly more common among isolates with ertapenem MICs of ≥0.125 mg/L than isolates with MICs of ≤0.094 mg/L (22.7% and 5.6%, respectively; χ2 = 13.815, P = 0.001). Ertapenem may be effective therapy for gonococcal isolates with decreased susceptibility or resistance to ESCs and isolates with identifiable genetic resistance determinants.

Microbial Lipid A Remodeling Controls Cross-Presentation Efficiency and CD8 T Cell Priming by Modulating Dendritic Cell Function.

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis, an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-ß) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis-associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders.

Susceptibility Trends of Zoliflodacin against Multidrug-Resistant Neisseria gonorrhoeae Clinical Isolates in Nanjing, China, 2014 to 2018.

Previously, we reported the potent activity of a novel spiropyrimidinetrione, zoliflodacin, against Neisseria gonorrhoeae isolates collected in 2013 from symptomatic men in Nanjing, China. Here, we investigated trends of susceptibilities to zoliflodacin in 986 isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in the gyrA, gyrB, parC, parE, and mtrR genes were determined by PCR and sequencing. The MICs of zoliflodacin ranged from ≤0.002 to 0.25 mg/liter; the overall MIC50 and MIC90 were 0.06 mg/liter and 0.125 mg/liter, respectively, in 2018, increasing 2-fold from 2014. However, the percentage of isolates with lower zoliflodacin MICs declined in each year sequentially, while the percentage with higher MICs increased yearly (P ≤ 0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥ 1 mg/liter); 21.2% (209/986) were resistant to azithromycin (≥1 mg/liter), 43.4% (428/986) were penicillinase-producing N. gonorrhoeae (PPNG), 26.9% (265/986) were tetracycline-resistant N. gonorrhoeae (TRNG), and 19.4% (191/986) were multidrug-resistant (MDR) isolates. 202 isolates with the lowest (≤0.002 to 0.015 mg/liter) and highest (0.125 to 0.25 mg/liter) zoliflodacin MICs were quinolone resistant with double or triple mutations in gyrA; 193/202 (95.5%) also had mutations in parC There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; an S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin continues to have excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin, and extended-spectrum cephalosporins.

Oral infection with a periodontal pathogen alters oral and gut microbiomes.

Periodontal disease, an inflammatory bone disease of the oral cavity, affects more than 50% of the United States population over the age of 30. The Gram-negative, anaerobic bacterium Porphyromonas gingivalis, the etiological agent of periodontal disease, is known to induce dysbiosis of the oral microbiome while promoting inflammatory bone loss. We have recently reported that P. gingivalis can also alter the gut microbiota of mice prone to develop inflammatory atherosclerosis. However, it is still unknown whether P. gingivalis induces similar changes to the gut microbiome as it does to oral microbiome. In this study, we demonstrate that P. gingivalis infection increases the diversity of the oral microbiome, allowing for colonization of potentially opportunistic species in the oral microbiome and overgrowth of commensal species in both the oral and gut microbiomes. Since periodontal disease treatment in humans typically involves antibiotic treatment, we also examined the combined effect of P. gingivalis infection on mice pretreated with oral antibiotics. By correlating the oral and cecal microbiota of P. gingivalis-infected mice fed a normal chow diet, we identified blooms of the Gram-negative genera Barnesiella and Bacteroides and imbalances of mucin-degrading bacteria. These disrupted community structures were predicted to have increased detrimental functional capacities including increased flavonoid degradation and l-histidine fermentation. Though antibiotic pretreatment (without P. gingivlais) had a dominant impact on the cecal microbiome, P. gingivalis infection of mice with or without antibiotic pretreatment increased the abundance of the phylum Firmicutes and the Porphyromonadaceae family in the cecum. Collectively, our study demonstrates that P. gingivalis oral infection disrupted the oral and cecal microbiomes of otherwise unperturbed mice, altering their community membership and functional potential.

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1ß (IL-1ß) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1ß production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Distinct lipid a moieties contribute to pathogen-induced site-specific vascular inflammation.

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE(-/-) mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE(-/-) mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

Neisseria gonorrhoeae Modulates Cell Death in Human Endocervical Epithelial Cells through Export of Exosome-Associated cIAP2.

Several bacterial pathogens persist and survive in the host by modulating host cell death pathways. We previously demonstrated that Neisseria gonorrhoeae, a Gram-negative pathogen responsible for the sexually transmitted infection gonorrhea, protects against exogenous induction of apoptosis in human cervical epithelial cells. However, induction of cell death by N. gonorrhoeae has also been reported in other cell types. The mechanisms by which N. gonorrhoeae modulates cell death are not clear, although a role for the inhibitor of apoptosis-2 (cIAP2) has been proposed. In this study, we confirmed that N. gonorrhoeae induces production of cIAP2 in human cervical epithelial cells. High levels of intracellular cIAP2 were detected early after N. gonorrhoeae stimulation, which was followed by a marked decrease at 24 h. At this time point, we observed increased levels of extracellular cIAP2 associated with exosomes and an overall increase in production of exosomes. Inhibition of cIAP2 in N. gonorrhoeae-stimulated epithelial cells resulted in increased cell death and interleukin-1ß (IL-1ß) production. Collectively these results indicate that N. gonorrhoeae stimulation of human endocervical epithelial cells induces the release of cIAP2, an essential regulator of cell death and immune signaling.

Interleukin 1 receptor 1 and interleukin 1ß regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans.

OBJECTIVE: Interleukin 1 Receptor 1 (IL1R1) and its ligand, IL1ß, are upregulated in cardiovascular disease, obesity, and infection. Previously, we reported a higher level of IL1R1 transcripts in platelets from obese individuals of the Framingham Heart Study (FHS), but its functional effect in platelets has never been described. Additionally, IL1ß levels are increased in atherosclerotic plaques and in bacterial infections. The aim of this work is to determine whether IL1ß, through IL1R1, can activate platelets and megakaryocytes to promote atherothrombosis. APPROACH AND RESULTS: We found that IL1ß-related genes from platelets, as measured in 1819 FHS participants, were associated with increased body mass index, and a direct relationship was shown in wild-type mice fed a high-fat diet. Mechanistically, IL1ß activated nuclear factor-κB and mitogen-activated protein kinase signaling pathways in megakaryocytes. IL1ß, through IL1R1, increased ploidy of megakaryocytes to 64+ N by 2-fold over control. IL1ß increased agonist-induced platelet aggregation by 1.2-fold with thrombin and 4.2-fold with collagen. IL1ß increased adhesion to both collagen and fibrinogen, and heterotypic aggregation by 1.9-fold over resting. High fat diet-enhanced platelet adhesion was absent in IL1R1(-/-) mice. Wild-type mice infected with Porphyromonas gingivalis had circulating heterotypic aggregates (1.5-fold more than control at 24 hours and 6.2-fold more at 6 weeks) that were absent in infected IL1R1(-/-) and IL1ß(-/-) mice. CONCLUSIONS: In summary, IL1R1- and IL1ß-related transcripts are elevated in the setting of obesity. IL1R1/IL1ß augment both megakaryocyte and platelet functions, thereby promoting a prothrombotic environment during infection and obesity; potentially contributing to the development of atherothrombotic disease.

Macrophage-specific TLR2 signaling mediates pathogen-induced TNF-dependent inflammatory oral bone loss.

Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf(-/-)) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.

Computational analysis of bacterial RNA-Seq data.

Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.

Distinct gene signatures in aortic tissue from ApoE-/- mice exposed to pathogens or Western diet.

BACKGROUND: Atherosclerosis is a progressive disease characterized by inflammation and accumulation of lipids in vascular tissue. Porphyromonas gingivalis (Pg) and Chlamydia pneumoniae (Cp) are associated with inflammatory atherosclerosis in humans. Similar to endogenous mediators arising from excessive dietary lipids, these Gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. In this study, we identified gene expression profiles that characterize P. gingivalis, C. pneumoniae, and Western diet (WD) at acute and chronic time points in aortas of Apolipoprotein E (ApoE-/-) mice. RESULTS: At the chronic time point, we observed that P. gingivalis was associated with a high number of unique differentially expressed genes compared to C. pneumoniae or WD. For the top 500 differentially expressed genes unique to each group, we observed a high percentage (76%) that exhibited decreased expression in P. gingivalis-treated mice in contrast to a high percentage (96%) that exhibited increased expression in WD mice. C. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. Gene Set Enrichment Analysis (GSEA) revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and PPAR pathways in response to P. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and PPAR pathways in response to C. pneumoniae; WD was associated with increased expression of immune and inflammatory pathways. DAVID analysis of gene clusters identified by two-way ANOVA at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in ApoE-/- mice; these changes were enhanced in P. gingivalis-treated mice but attenuated in C. pneumoniae-treated mice. Notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli. CONCLUSIONS: Despite the common outcome of P. gingivalis, C. pneumoniae, and WD on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified. Our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways.

Microbial carriage state of peripheral blood dendritic cells (DCs) in chronic periodontitis influences DC differentiation, atherogenic potential.

The low-grade oral infection chronic periodontitis (CP) has been implicated in coronary artery disease risk, but the mechanisms are unclear. In this study, a pathophysiological role for blood dendritic cells (DCs) in systemic dissemination of oral mucosal pathogens to atherosclerotic plaques was investigated in humans. The frequency and microbiome of CD19(-)BDCA-1(+)DC-SIGN(+) blood myeloid DCs (mDCs) were analyzed in CP subjects with or without existing acute coronary syndrome and in healthy controls. FACS analysis revealed a significant increase in blood mDCs in the following order: healthy controls < CP < acute coronary syndrome/CP. Analysis of the blood mDC microbiome by 16S rDNA sequencing showed Porphyromonas gingivalis and other species, including (cultivable) Burkholderia cepacia. The mDC carriage rate with P. gingivalis correlated with oral carriage rate and with serologic exposure to P. gingivalis in CP subjects. Intervention (local debridement) to elicit a bacteremia increased the mDC carriage rate and frequency in vivo. In vitro studies established that P. gingivalis enhanced by 28% the differentiation of monocytes into immature mDCs; moreover, mDCs secreted high levels of matrix metalloproteinase-9 and upregulated C1q, heat shock protein 60, heat shock protein 70, CCR2, and CXCL16 transcripts in response to P. gingivalis in a fimbriae-dependent manner. Moreover, the survival of the anaerobe P. gingivalis under aerobic conditions was enhanced when within mDCs. Immunofluorescence analysis of oral mucosa and atherosclerotic plaques demonstrate infiltration with mDCs, colocalized with P. gingivalis. Our results suggest a role for blood mDCs in harboring and disseminating pathogens from oral mucosa to atherosclerosis plaques, which may provide key signals for mDC differentiation and atherogenic conversion.

Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.

Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via TLRs link stimulation by multiple pathogens to atherosclerosis. However, how pathogen-specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood. In this study, atherosclerosis-prone apolipoprotein-E null (ApoE(-/-)) and TLR4-deficient (ApoE(-/-)TLR4(-/-)) mice were orally infected with the periodontal pathogen Porphyromonas gingivalis. ApoE(-/-)TLR4(-/-) mice were markedly more susceptible to atherosclerosis after oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE(-/-)TLR4(-/-) mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of Th1 immunity and regulatory T cell infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell IL-12 and IL-10, which are prototypic Th1 and regulatory T cell polarizing cytokines. We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.

Neisseria prophage repressor implicated in gonococcal pathogenesis.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect and colonize multiple mucosal sites in both men and women. The ability to cope with different environmental conditions requires tight regulation of gene expression. In this study, we identified and characterized a gonococcal transcriptional regulatory protein (Neisseria phage repressor [Npr]) that was previously annotated as a putative gonococcal phage repressor protein. Npr was found to repress transcription of NGNG_00460 to NGNG_00463 (NGNG_00460-00463), an operon present within the phage locus NgoΦ4. Npr binding sites within the NGNG_00460-00463 promoter region were found to overlap the -10 and -35 promoter motifs. A gonococcal npr mutant demonstrated increased adherence to and invasion of human endocervical epithelial cells compared to a wild-type gonococcal strain. Likewise, the gonococcal npr mutant exhibited enhanced colonization in a gonococcal mouse model of mucosal infection. Analysis of the gonococcal npr mutant using RNA sequence (RNA-seq) analysis demonstrated that the Npr regulon is limited to the operon present within the phage locus. Collectively, our studies have defined a new gonococcal phage repressor protein that controls the transcription of genes implicated in gonococcal pathogenesis.

Disruption of immune regulation by microbial pathogens and resulting chronic inflammation.

Activation of the immune response is a tightly regulated, coordinated effort that functions to control and eradicate exogenous microorganisms, while also responding to endogenous ligands. Determining the proper balance of inflammation is essential, as chronic inflammation leads to a wide array of host pathologies. Bacterial pathogens can instigate chronic inflammation via an extensive repertoire of evolved evasion strategies that perturb immune regulation. In this review, we discuss two model pathogens, Mycobacterium tuberculosis and Porphyromonas gingivalis, which efficiently escape various aspects of the immune system within professional and non-professional immune cell types to establish chronic inflammation.

Increase in Multidrug Resistant Neisseria gonorrhoeae FC428-Like Isolates Harboring the Mosaic penA 60.001 Gene, in Nanjing, China (2017-2020).

Background: Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China. Objective: To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features. Methods: N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS). Results: Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city's N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates. Conclusion: penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.

In Vitro Pre-Clinical Evaluation of a Gonococcal Trivalent Candidate Vaccine Identified by Transcriptomics.

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, poses a significant global public health threat. Infection in women can be asymptomatic and may result in severe reproductive complications. Escalating antibiotic resistance underscores the need for an effective vaccine. Approaches being explored include subunit vaccines and outer membrane vesicles (OMVs), but an ideal candidate remains elusive. Meningococcal OMV-based vaccines have been associated with reduced rates of gonorrhea in retrospective epidemiologic studies, and with accelerated gonococcal clearance in mouse vaginal colonization models. Cross-protection is attributed to shared antigens and possibly cross-reactive, bactericidal antibodies. Using a Candidate Antigen Selection Strategy (CASS) based on the gonococcal transcriptome during human mucosal infection, we identified new potential vaccine targets that, when used to immunize mice, induced the production of antibodies with bactericidal activity against N. gonorrhoeae strains. The current study determined antigen recognition by human sera from N. gonorrhoeae-infected subjects, evaluated their potential as a multi-antigen (combination) vaccine in mice and examined the impact of different adjuvants (Alum or Alum+MPLA) on functional antibody responses to N. gonorrhoeae. Our results indicated that a stronger Th1 immune response component induced by Alum+MPLA led to antibodies with improved bactericidal activity. In conclusion, a combination of CASS-derived antigens may be promising for developing effective gonococcal vaccines.

Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase.

Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam(3)CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin alpha(IIb)beta(3), generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam(3)CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

Diet-Induced Non-alcoholic Fatty Liver Disease and Associated Gut Dysbiosis Are Exacerbated by Oral Infection.

Increasing evidence indicates that chronic inflammation due to periodontal disease is associated with progression of non-alcoholic fatty liver disease (NAFLD) caused by a Western diet. NAFLD has also been associated with oral infection with the etiological agent of periodontal disease, Porphyromonas gingivalis. P. gingivalis oral infection has been shown to induce cardiometabolic disease features including hepatic lipid accumulation while also leading to dysbiosis of the gut microbiome. However, the impact of P. gingivalis infection on the gut microbiota of mice with diet-induced NAFLD and the potential for those changes to mediate NAFLD progression has yet to be determined. In the current study, we have demonstrated that P. gingivalis infection induced sustained alterations of the gut microbiota composition and predicted functions, which was associated with the promotion of NAFLD in steatotic mice. Reduced abundance of short-chain fatty acid-producing microbiota was observed after both acute and chronic P. gingivalis infection. Collectively, our findings demonstrate that P. gingivalis infection produces a persistent change in the gut microbiota composition and predicted functions that promotes steatosis and metabolic disease.