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Chlamydia trachomatis is an obligate intracellular bacterium that causes serious diseases in humans. Rectal infection and disease caused by this pathogen are important yet understudied aspects of C. trachomatis natural history. The University of Washington Chlamydia Repository has a large collection of male-rectal-sourced strains (MSM rectal strains) isolated in Seattle, USA and Lima, Peru. Initial characterization of strains collected over 30 years in both Seattle and Lima led to an association of serovars G and J with male rectal infections. Serovar D, E, and F strains were also collected from MSM patients. Genome sequence analysis of a subset of MSM rectal strains identified a clade of serovar G and J strains that had high overall genomic identity. A genome-wide association study was then used to identify genomic loci that were correlated with tissue tropism in a collection of serovar-matched male rectal and female cervical strains. The polymorphic membrane protein PmpE had the strongest correlation, and amino acid sequence alignments identified a set of PmpE variable regions (VRs) that were correlated with host or tissue tropism. Examination of the positions of VRs by the protein structure-predicting Alphafold2 algorithm demonstrated that the VRs were often present in predicted surface-exposed loops in both PmpE and PmpH protein structure. Collectively, these studies identify possible tropism-predictive loci for MSM rectal C. trachomatis infections and identify predicted surface-exposed variable regions of Pmp proteins that may function in MSM rectal versus cervical tropism differences.

The Chlamydiae are obligate intracellular pathogens that develop and multiply within a poorly characterized parasitophorous vacuole (the inclusion) during growth. Chlamydia abortus is a major pathogen of sheep and other ruminants, and its inclusion development is poorly characterized. We used immunofluorescence microscopy, quantitative culture, and qPCR to examine C. abortus inclusion development and to examine the interaction of C. abortus inclusions with those formed by other species. Antibodies used in these studies include sera from ewes from production facilities that were naturally infected with C. abortus. Multiple inclusions are often found in C. abortus-infected cells, even in populations infected at very low multiplicity of infection. Labeling of fixed cells with sera from infected sheep revealed fibrous structures that extend away from the inclusion into the cytoplasm of the host cell. C. abortus inclusions fused with C. caviae and C. psittaci inclusions in coinfected cells. Inclusions formed by C. abortus and C. caviae did not fuse with inclusions formed by C. trachomatis, C. pneumoniae, or C. pecorum. The ability of inclusions to fuse was correlated with the overall genomic relatedness between species, and with sequence similarity in the inclusion membrane protein IncA. Quantitative PCR data demonstrated that C. abortus grows at a decreased rate during coinfections with C. caviae, while C. caviae growth was unaffected. The collected data add depth to our understanding of inclusion development in this significant zoonotic veterinary pathogen.

Lateral gene transfer (LGT) among Chlamydia trachomatis strains is common, in both isolates generated in the laboratory and those examined directly from patients. In contrast, there are very few examples of recent acquisition of DNA by any Chlamydia spp. from any other species. Interspecies LGT in this system was analyzed using crosses of tetracycline (Tc)-resistant C. trachomatis L2/434 and chloramphenicol (Cam)-resistant C. muridarum VR-123. Parental C. muridarum strains were created using a plasmid-based Himar transposition system, which led to integration of the Camr marker randomly across the chromosome. Fragments encompassing 79% of the C. muridarum chromosome were introduced into a C. trachomatis background, with the total coverage contained on 142 independent recombinant clones. Genome sequence analysis of progeny strains identified candidate recombination hot spots, a property not consistent with in vitroC. trachomatis × C. trachomatis (intraspecies) crosses. In both interspecies and intraspecies crosses, there were examples of duplications, mosaic recombination endpoints, and recombined sequences that were not linked to the selection marker. Quantitative analysis of the distribution and constitution of inserted sequences indicated that there are different constraints on interspecies LGT than on intraspecies crosses. These constraints may help explain why there is so little evidence of interspecies genetic exchange in this system, which is in contrast to very widespread intraspecies exchange in C. trachomatis. IMPORTANCE Genome sequence analysis has demonstrated that there is widespread lateral gene transfer among strains within the species C. trachomatis and with other closely related Chlamydia species in laboratory experiments. This is in contrast to the complete absence of foreign DNA in the genomes of sequenced clinical C. trachomatis strains. There is no understanding of any mechanisms of genetic transfer in this important group of pathogens. In this report, we demonstrate that interspecies genetic exchange can occur but that the nature of the fragments exchanged is different than those observed in intraspecies crosses. We also generated a large hybrid strain library that can be exploited to examine important aspects of chlamydial disease.