Chlamydiae growing in target mucosal human epithelial cells in vitro can transition from their normal developmental cycle progression -- alternating between infectious but metabolically-inactive elementary bodies (EB) to metabolically-active but non-infectious reticulate bodies (RB) and back to EB -- into a state of “persistence”. Persistence in vitro is defined as “viable but non-cultivable chlamydiae” involving “morphologically enlarged, aberrant, non-dividing RB”. The condition is “reversible” to yield infectious EB on removal of the inducers, including penicillin, interferon-gamma, iron/nutrient starvation, concomitant herpes infection, or maturation of the host cell into its physiologically-differentiated state. All aberrant RB phenotypes are not the same due to differing up/down regulated chlamydial gene sets and subsequent host responses. While all persistence-inducing conditions exist in vivo, key questions include whether or not (i) aberrant chlamydial RB occur in vivo during the alternating acute-silent chronic-acute chlamydial infection scenario that exists in infected patients and animals and (ii) if such aberrant RB can contribute to prolonged, chronic inflammation, fibrosis and scarring .

IFNβ has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during chlamydial infection. We determined that TREX-1, a host 3’to 5’ exonuclease, reduced IFNβ expression significantly during chlamydial infection using siRNA and gene knock out fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cGAS has been shown to bind cytosolic DNA to generate cGAMP, which binds to the signaling adaptor STING to induce IFNβ expression. We determined that cGAS is required for IFNβ expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFNβ, co-culture of cells depleted for either STING or cGAS rescued IFNβ expression. These data demonstrate that cGAMP produced in infected cGAS+STING− cells can migrate into adjacent cells via gap junctions to function in trans in cGAS−STING+ cells. Further, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggests that effectors from infected cells can directly upregulate IFNβ expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis.

SummaryEpidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo . We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in coinfected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 coinfection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL , ftsK , ftsW , dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but noncultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo , these results also suggest that disease severity might be increased in coinfected individuals.

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Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxE HP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptA HP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxE HP -deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxE HP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxE HP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxE HP . Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 g/ml). However, disruption of lpxE HP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 g/ml). In conclusion, we have characterized the first gram-negative LpxEdeficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.