Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

Comprehensive genome analysis and comparisons of the swine pathogen, Chlamydia suis reveals unique ORFs and candidate host-specificity factors.

Chlamydia suis, a ubiquitous swine pathogen, has the potential for zoonotic transmission to humans and often encodes for resistance to the primary treatment antibiotic, tetracycline. Because of this emerging threat, comparative genomics for swine isolate R19 with inter- and intra-species genomes was performed. A 1.094 Mb genome was determined through de novo assembly of Illumina high throughput sequencing reads. Annotation and subsystem analyses were conducted, revealing 986 putative genes (Chls_###) that are predominantly orthologs to other known Chlamydia genes. Subsequent comparative genomics revealed a high level of genomic synteny and overall sequence identity with other Chlamydia while 92 unique C. suis open reading frames were annotated. Direct comparison of Chlamydia-specific gene families that included the plasticity zone, inclusion membrane proteins, polymorphic membrane proteins and the major outer membrane protein, demonstrated high gene content identity with C. trachomatis and C. muridarum. These comparisons also identified diverse components that potentially could contribute to host-specificity. This study constitutes the first genome-wide comparative analysis for C. suis, generating a fully annotated reference genome. These studies will enable focused efforts on factors that provide key species specificity and adaptation to cognate hosts that are attributed to chlamydial infections, including humans.

Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis support a role in TCA cycle regulation.

Chlamydia trachomatis is an obligate intracellular bacteria that undergo dynamic morphologic and physiologic conversions upon gaining an access to a eukaryotic cell. These conversions likely require the detection of key environmental conditions and regulation of metabolic activity. Chlamydia encodes homologs to proteins in the Rsb phosphoregulatory partner-switching pathway, best described in Bacillus subtilis. ORF CT588 has a strong sequence similarity to RsbU cytoplasmic phosphatase domain but also contains a unique periplasmic sensor domain that is expected to control the phosphatase activity. A 1.7 Å crystal structure of the periplasmic domain of the RsbU protein from C. trachomatis (PDB 6MAB) displays close structural similarity to DctB from Vibrio and Sinorhizobium. DctB has been shown, both structurally and functionally, to specifically bind to the tricarboxylic acid (TCA) cycle intermediate succinate. Surface plasmon resonance and differential scanning fluorimetry of TCA intermediates and potential metabolites from a virtual screen of RsbU revealed that alpha-ketoglutarate, malate and oxaloacetate bound to the RsbU periplasmic domain. Substitutions in the putative binding site resulted in reduced binding capabilities. An RsbU null mutant showed severe growth defects which could be restored through genetic complementation. Chemical inhibition of ATP synthesis by oxidative phosphorylation phenocopied the growth defect observed in the RsbU null strain. Altogether, these data support a model with the Rsb system responding differentially to TCA cycle intermediates to regulate metabolism and key differentiation processes.

Development of Transposon Mutagenesis for Chlamydia muridarum.

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

Chromosomal Recombination Targets in Chlamydia Interspecies Lateral Gene Transfer.

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. trachomatisIMPORTANCE 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.

Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer.

Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.IMPORTANCEChlamydia trachomatis infections have an immense impact on public health; however, understanding the basic biology and pathogenesis of this organism has been stalled by the limited repertoire of genetic tools. This report describes the successful adaptation of an important tool that has been lacking in Chlamydia studies: transposon mutagenesis. This advance enabled the generation of 105 insertional mutants, demonstrating that numerous gene products are not essential for in vitro growth. Mammalian infections using these mutants revealed that several gene products are important for infections in vivo Moreover, this tool enabled the investigation and discovery of a gene critical for lateral gene transfer; a process fundamental to the evolution of bacteria and likely for Chlamydia as well. The development of transposon mutagenesis for Chlamydia has broad impact for the field and for the discovery of genes associated with selected phenotypes, providing an additional avenue for the discovery of molecular mechanisms used for pathogenesis and for a more thorough understanding of this important pathogen.

Demonstration of Persistent Infections and Genome Stability by Whole-Genome Sequencing of Repeat-Positive, Same-Serovar Chlamydia trachomatis Collected From the Female Genital Tract.

Background: The biology of recurrent or long-term infections of humans by Chlamydia trachomatis is poorly understood. Because repeated or persistent infections are correlated with serious complications in humans, understanding these processes may improve clinical management and public health disease control. Methods: We conducted whole-genome sequence analysis on C. trachomatis isolates collected from a previously described patient set in which individuals were shown to be infected with a single serovar over a lengthy period. Results: Data from 5 of 7 patients showed compelling evidence for the ability of these patients to harbor the same strain for 3-5 years. Mutations in these strains were cumulative, very uncommon, and not linked to any single protein or pathway. Serovar J strains isolated from 1 patient 3 years apart did not accumulate a single base change across the genome. In contrast, the sequence results of 2 patients, each infected only with serovar Ia strains, revealed multiple same-serovar infections over 1-5 years. Conclusions: These data demonstrate examples of long-term persistence in patients in the face of repeated antibiotic therapy and show that pathogen mutational strategies are not important in persistence of this pathogen in patients.