Laszlo Kari

The Chlamydia trachomatis plasmid is a transcriptional regulator of chromosomal genes and a virulence factor.

ABSTRACT Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatis strain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatis strains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 × 106 inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 × 104 IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmids ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

Chlamydia trachomatis Plasmid-Encoded Pgp4 Is a Transcriptional Regulator of Virulence-Associated Genes

ABSTRACT Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmids role in chlamydial pathogenesis at the molecular level.

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.

Pathogenic Diversity among Chlamydia trachomatis Ocular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. To assess the role played by pathogen variation in disease outcome, we analyzed the genomes of 4 trachoma strains representative of the 3 major trachoma serotypes, using microarray-based comparative genome sequencing. Outside of ompA, trachoma strains differed primarily in a very small subset of genes (n = 22). These subtle genetic variations were manifested in profound differences in virulence as measured by in vitro growth rate, burst size, plaque morphology, and interferon-gamma sensitivity but most importantly in virulence as shown by ocular infection of nonhuman primates. Our findings are the first to identify genes that correlate with differences in pathogenicity among trachoma strains.

Chlamydia trachomatis Native Major Outer Membrane Protein Induces Partial Protection in Nonhuman Primates: Implication for a Trachoma Transmission-Blocking Vaccine

A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized monkeys produced a broadly cross-reactive, Ag-specific IFN-γ response equivalent to that induced by experimental infection. Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3–14). However, at later time points, they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.

Frameshift Mutations in a Single Novel Virulence Factor Alter the In Vivo Pathogenicity of Chlamydia trachomatis for the Female Murine Genital Tract

ABSTRACT Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.

Coordinate control of cell cycle regulatory genes in zebrafish development tested by cyclin D1 knockdown with morpholino phosphorodiamidates and hydroxyprolyl-phosphono peptide nucleic acids

During early zebrafish (Danio rerio) development zygotic transcription does not begin until the mid-blastula transition (MBT) ∼3 h after fertilization. MBT demarcates transition from synchronous short cell cycles of S and M phases exclusively to full cycles encompassing G1 and G2 phases. Transcriptional profiling and RT–PCR analyses during these phases enabled us to determine that this shift corresponds to decreased transcript levels of S/M phase cell cycle control genes (e.g. ccna2, ccnb1, ccnb2 and ccne) and increased transcript levels of ccnd1, encoding cyclin D1, and orthologs of p21 (p21-like) and retinoblastoma (Rb-like 1). To investigate the regulation of this process further, the translation of ccnd1 mRNA, a G1/S checkpoint control element, was impaired by microinjection of ccnd1-specific morpholino phosphorodiamidate (MO) 20mer or hydroxyprolyl-phosphono peptide nucleic acid (HypNA-pPNA) 16mer antisense oligonucleotides. The resulting downregulation of cyclin D1 protein resulted in microophthalmia and microcephaly, but not lethality. The phenotypes were not seen with 3-mismatch MO 20mers or 1-mismatch HypNA-pPNA 16mers, and were rescued by an exogenous ccnd1 mRNA construct with five mismatches. Collectively, these results indicate that transcription of key molecular determinants of asynchronous cell cycle control in zebrafish embryos commences at MBT and that the reduction of cyclin D1 expression compromises zebrafish eye and head development.

CD8+ T Cells Define an Unexpected Role in Live-Attenuated Vaccine Protective Immunity against Chlamydia trachomatis Infection in Macaques

Trachoma, caused by the obligate intracellular organism Chlamydia trachomatis, is the world’s leading cause of preventable blindness for which a vaccine is needed. We have previously shown that a plasmid-deficient live-attenuated trachoma vaccine delivered ocularly to macaques elicited either solid or partial protective immunity against a virulent ocular challenge. Solidly protected macaques shared the same MHC class II alleles implicating CD4+ T cells in superior protective immunity. Understandably, we sought to define T cell immune correlates in these animals to potentially improve vaccine efficacy. In this study, following a 2-y resting period, these macaques were boosted i.m. with the live-attenuated trachoma vaccine and their peripheral T cell anamnestic responses studied. Both solidly and partially protected macaques exhibited a CD4+ and CD8+ T cell anamnestic response following booster immunization. CD8+ but not CD4+ T cells from solidly protected macaques proliferated against soluble chlamydial Ag. We observed a more rapid T cell inflammatory cytokine response in tears of solidly protected animals following ocular rechallenge. Most notably, depletion of CD8+ T cells in solidly protected macaques completely abrogated protective immunity. Collectively, our findings support the conclusion that CD8+ T cells play an important but unexpected role in live-attenuated trachoma vaccine-mediated protective immunity.

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

ABSTRACT Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.

Chlamydia trachomatis Polymorphic Membrane Protein D Is a Virulence Factor Involved in Early Host-Cell Interactions

ABSTRACT Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The C. trachomatis polymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a pmpD null mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that pmpD is not an essential chlamydial gene and the pmpD null mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the pmpD null mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in pmpD null infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a C. trachomatis gene to disease pathogenesis.