Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Chlamydia trachomatis Plasmid Gene Protein 3 Is Essential for the Establishment of Persistent Infection and Associated Immunopathology.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease afflicting hundreds of millions of people globally. A fundamental but poorly understood pathophysiological characteristic of chlamydial infection is the propensity to cause persistent infection that drives damaging inflammatory disease. The chlamydial plasmid is a virulence factor, but its role in the pathogenesis of persistent infection capable of driving immunopathology is unknown. Here, we show by using mouse and nonhuman primate infection models that the secreted plasmid gene protein 3 (Pgp3) is essential for establishing persistent infection. Ppg3-dependent persistent genital tract infection resulted in a severe endometritis caused by an intense infiltration of endometrial submucosal macrophages. Pgp3 released from the cytosol of lysed infected oviduct epithelial cells, not organism outer membrane-associated Pgp3, inhibited the chlamydial killing activity of antimicrobial peptides. Genetic Pgp3 rescue experiments in cathelin-related antimicrobial peptide (CRAMP)-deficient mice showed Pgp3-targeted antimicrobial peptides to subvert innate immunity as a pathogenic strategy to establish persistent infection. These findings provide important advances in understanding the role of Pgp3 in the pathogenesis of persistent chlamydial infection and associated immunopathology.IMPORTANCEChlamydia trachomatis can cause persistent infection that drives damaging inflammatory responses resulting in infertility and blindness. Little is known about chlamydial genes that cause persistence or factors that drive damaging pathology. In this work, we show that the C. trachomatis plasmid protein gene 3 (Pgp3) is the essential virulence factor for establishing persistent female genital tract infection and provide supportive evidence that Pgp3 functions similarly in a nonhuman primate trachoma model. We further show that persistent Ppg3-dependent infection drives damaging immunopathology. These results are important advances in understanding the pathophysiology of chlamydial persistence.

Chlamydia trachomatis Lipopolysaccharide Evades the Canonical and Noncanonical Inflammatory Pathways To Subvert Innate Immunity.

Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections. C. trachomatis sexually transmitted infections are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we studied the ability of chlamydial LPS to activate the proinflammatory canonical and noncanonical inflammasome pathways in mouse bone marrow-derived macrophages (BMDM). We show, in comparison to Escherichiacoli LPS, that C. trachomatis LPS-treated BMDM produce significantly less IL-6, TNF, and type I interferon mRNA, indicating that downstream signaling through the canonical TLR4 myddosome and triffosome pathways was blocked. We confirmed this in C. trachomatis LPS-treated BMDM by showing the lack of NF-κB and IRF3 phosphorylation, respectively. Interestingly, C. trachomatis LPS bound CD14 and promoted its endocytosis; however; it did not promote efficient TLR4/MD-2 dimerization or endocytosis, known requirements for myddosome and triffosome signaling pathways. We further found that transfection of BMDM with C. trachomatis LPS did not cause pyroptotic cell ballooning, cytotoxicity, or IL-1ß secretion, all characteristic features of noncanonical inflammasome activation. Western blotting confirmed that cytosolic C. trachomatis LPS failed to signal through caspase-11, as shown by the lack of gasdermin D, caspase-1, or IL-1ß proteolytic cleavage. We propose that chlamydiae evolved a unique LPS structure as a pathogenic strategy to avoid canonical and noncanonical innate immune signaling and conclude that this strategy might explain the high incidence of asymptomatic infections.IMPORTANCEChlamydia trachomatis is the most common bacterial cause of sexually transmitted infections (STI). C. trachomatis STI are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we found that C. trachomatis LPS is not capable of engaging the canonical TLR4/MD-2 or noncanonical caspase-11 inflammatory pathways. The inability of C. trachomatis LPS to trigger innate immunity inflammatory pathways is related to its unique fatty acid structure. Evolutionary modification of the LPS structure likely evolved as a pathogenic strategy to silence innate host defense mechanisms. The findings might explain the high incidence of asymptomatic chlamydial genital infection.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

Generation of targeted Chlamydia trachomatis null mutants.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells.

Gamma interferon (IFN-gamma)-induced effector mechanisms have potent antichlamydial activities that are critical to host defense. The most prominent and well-studied effectors are indoleamine dioxygenase (IDO) and nitric oxide (NO) synthase. The relative contributions of these mechanisms as inhibitors of chlamydial in vitro growth have been extensively studied using different host cells, induction mechanisms, and chlamydial strains with conflicting results. Here, we have undertaken a comparative analysis of cytokine- and lipopolysaccharide (LPS)-induced IDO and NO using an extensive assortment of human and murine host cells infected with human and murine chlamydial strains. Following cytokine (IFN-gamma or tumor necrosis factor alpha) and/or LPS treatment, the majority of human cell lines induced IDO but failed to produce NO. Conversely, the majority of mouse cell lines studied produced NO, not IDO. Induction of IDO in human cell lines inhibited growth of L2 and mouse pneumonitis agent, now referred to as Chlamydia muridarum MoPn equally in all but two lines, and inhibition was completely reversible by the addition of tryptophan. IFN-gamma treatment of mouse cell lines resulted in substantially greater reduction of L2 than MoPn growth. However, despite elevated NO production by murine cells, blockage of NO synthesis with the l-arginine analogue N-monomethyl-l-arginine only partially rescued chlamydial growth, suggesting the presence of another IFN-gamma-inducible antichlamydial mechanism unique to murine cells. Moreover, NO generated from the chemical nitric oxide donor sodium nitroprusside showed little direct effect on chlamydial infectivity or growth, indicating a natural resistance to NO. Finally, IFN-gamma-inducible IDO expression in human HeLa cells was inhibited following exogenous NO treatment, resulting in a permissive environment for chlamydial growth. In summary, cytokine- and LPS-inducible effectors produced by human and mouse cells differ and, importantly, these host-specific effector responses result in chlamydial strain-specific antimicrobial activities.

Chlamydial IFN-gamma immune evasion is linked to host infection tropism.

Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host-pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host-pathogen niche revolves around a restricted repertoire of host species-specific IFN-gamma-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-gamma induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-gamma induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (Iigp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-gamma-mediated immunity.

C. trachomatis-infection accelerates metabolism of phosphatidylcholine derived from low density lipoprotein but does not affect phosphatidylcholine secretion from hepatocytes.

BACKGROUND: Chlamydia trachomatis is a prevalent sexually transmitted disease and the leading cause of infectious blindness in developing nations. It was not known if C. trachomatis-infection influenced metabolism of lipoprotein-derived phospholipids. Nor was it known if C. trachomatis-infection altered phosphatidylcholine (PC) secretion from hepatocytes. In the current study, low density lipoprotein (LDL)-derived [methyl-3H]PC metabolism was examined in L929 cells infected with C. trachomatis to determine if PC derived from LDL could serve as a potential source of PC trafficked to C. trachomatis. In addition, release of endogenously synthesized [methyl-3H]PC into the medium was examined in rat liver hepatocytes infected with C. trachomatis to determine if infection altered PC secretion. RESULTS: L929 cells 20 h post infection exhibited a 39% (p < 0.05) reduction in radioactivity in PC but total radioactivity incorporation was unaltered compared to controls. Lysophosphatidyl [methyl-3H]choline (LPC) and aqueous [methyl-3H]choline metabolites were elevated 3.6-fold (p < 0.05) and 16.5-fold (p < 0.05), respectively, in C. trachomatis-infected cells and this was due to a 51% increase (p < 0.05) in calcium-dependent phospholipase A2 activity. Hepatocytes 22 h post infection then incubated for 16 h with [methyl-3H]choline showed elevated [methyl-3H]PC biosynthesis but [methyl-3H]PC secreted into the medium was unaltered compared to controls. In contrast, both cellular and medium lyso [methyl-3H]PC were elevated in C. trachomatis-infected cells. CONCLUSION: This study is the first to show that metabolism of LDL-derived PC is accelerated in C. trachomatis infection and to support the notion that LDL-delivered PC may potentially serve as a source of PC trafficked to Chlamydia. In addition, C. trachomatis-infection does not inhibit PC secretion from hepatocytes indicating that the pool of newly synthesized PC destined for lipoprotein secretion may differ from the pool of PC used for C. trachomatis membrane biosynthesis.

Activation of Raf/MEK/ERK/cPLA2 signaling pathway is essential for chlamydial acquisition of host glycerophospholipids.

Chlamydiae, a diverse group of obligate intracellular pathogens replicating within cytoplasmic vacuoles of eukaryotic cells, are able to acquire lipids from host cells. Here we report that activation of the host Raf-MEK-ERK-cPLA2 signaling cascade is required for the chlamydial uptake of host glycerophospholipids. Both the MAP kinase pathway (Ras/Raf/MEK/ERK) and Ca(2+)-dependent cytosolic phospholipase A2 (cPLA2) were activated in chlamydia-infected cells. The inhibition of cPLA2 activity resulted in the blockade of the chlamydial uptake of host glycerophospholipids and impairment in chlamydial growth. Blocking either c-Raf-1 or MEK1/2 activity prevented the chlamydial activation of ERK1/2, leading to the suppression of both chlamydial activation of the host cPLA2 and uptake of glycerophospholipids from the host cells. The chlamydia-induced phosphorylation of cPLA2 was also blocked by a dominant negative ERK2. Furthermore, activation of both ERK1/2 and cPLA2 was dependent on chlamydial growth and restricted within chlamydia-infected cells, suggesting an active manipulation of the host ERK-cPLA2 signaling pathway by chlamydiae.