HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Molecular Dynamics and Mode of Transmission of Koala Retrovirus as It Invades and Spreads through a Wild Queensland Koala Population

The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV: KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes require population level studies of their prevalence and diversity, especially when coinfected in the same population, and investigations of their modes of transmission in the wild. Toward this end, we studied a wild Queensland koala population of 290 animals over a 5-year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope protein sequence. In contrast, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B-positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B-positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data. IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B-infected/coinfected animals and the linkage of this infection to chlamydial disease, neoplasia, viral evolution, and spread.

CtHtrA: the lynchpin of the chlamydial surface and a promising therapeutic target.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection worldwide and the leading cause of preventable blindness. Reports have emerged of treatment failure, suggesting a need to develop new antibiotics to battle Chlamydia infection. One possible candidate for a new treatment is the protease inhibitor JO146, which is an effective anti-Chlamydia agent that targets the CtHtrA protein. CtHtrA is a lynchpin on the chlamydial cell surface due to its essential and multifunctional roles in the bacteria's stress response, replicative phase of development, virulence and outer-membrane protein assembly. This review summarizes the current understanding of CtHtrA function and presents a mechanistic model that highlights CtHtrA as an effective target for anti-Chlamydia drug development.

In vitro susceptibility of recent Chlamydia trachomatis clinical isolates to the CtHtrA inhibitor JO146.

The present study aimed to establish if a previously identified Chlamydia trachomatis HtrA (CtHtrA) inhibitor, JO146, is effective against currently circulating clinical isolates to validate if CtHtrA is a clinically relevant target for future therapeutic development. Inhibition of CtHtrA during the middle of the chlamydial replicative cycle until the completion of the cycle resulted in loss of infectious progeny for six unique clinical isolates representing different serovars. This supports the potential for CtHtrA to be a clinically relevant target for development of new therapeutics and suggests the importance of further investigation of JO146 as a lead compound.

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis.

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.

Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica.

OBJECTIVE: To develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Entamoeba histolytica E. histolytica, the causative agent of amebiasis. METHODS: The LAMP primer set was designed from E. histolytica hemolysin gene HLY6. Genomic DNA of E. histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions. Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye. RESULTS: Positive LAMP reactions turned turbid while negative ones remained clear. Upon addition of a fluorescent dye, all positive reactions turned green while the negative control remained orange under ambient light. After electrophoresis in 1.5% agarose gels, a ladder of multiple bands of different sizes can be observed in positive samples while no bands were detected in the negative control. The sensitivity of the assay was found to be 5 parasites per reaction which corresponds to approximately 15.8 ng/µ L DNA. The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species, Entamoeba dispar 39, and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used. CONCLUSIONS: The LAMP assay we have developed enables the detection of E. histolytica with rapidity and ease, therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

The protease inhibitor JO146 demonstrates a critical role for CtHtrA for Chlamydia trachomatis reversion from penicillin persistence.

The Chlamydia trachomatis serine protease HtrA (CtHtrA) has recently been demonstrated to be essential during the replicative phase of the chlamydial developmental cycle. A chemical inhibition strategy (serine protease inhibitor JO146) was used to demonstrate this essential role and it was found that the chlamydial inclusions diminish in size and are lost from the cell after CtHtrA inhibition without formation of viable elementary bodies. The inhibitor (JO146) was used in this study to investigate the role of CtHtrA for penicillin persistence and heat stress conditions for Chlamydia trachomatis. JO146 addition during penicillin persistence resulted in only minor reductions (~1 log) in the final viable infectious yield after persistent Chlamydia were reverted from persistence. However, JO146 treatment during the reversion and recovery from penicillin persistence was completely lethal for Chlamydia trachomatis. JO146 was completely lethal when added either during heat stress conditions, or during the recovery from heat stress conditions. These data together indicate that CtHtrA has essential roles during some stress environments (heat shock), recovery from stress environments (heat shock and penicillin persistence), as well as the previously characterized essential role during the replicative phase of the chlamydial developmental cycle. Thus, CtHtrA is an essential protease with both replicative phase and stress condition functions for Chlamydia trachomatis.

Profiles of Entamoeba histolytica-specific immunoglobulins in human sera.

OBJECTIVE: To determine the profiles of anti-Entamoeba histolytica (E. histolytica) IgA, IgG, and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens. METHODS: Serum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA, IgG, and IgM profiling through enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblot. RESULTS: ELISA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E. histolytica-specific IgG compared to IgA and IgM. Flow cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E. histolytica cells compared to non-diarrheic samples. Immunoreactive E. histolytica proteins with molecular weights ranging between 7 kDa and 292 kDa were recognized by diarrheic serum IgG, and 170 kDa and 250 kDa by non-diarrheic serum IgG. CONCLUSIONS: Our findings suggest that serum anti-E. histolytica IgG, compared with serum anti-E. histolytica IgA and IgM responses, was generally high in both diarrheic and non-diarrheic sera, indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers, respectively. In addition, serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E. histolytica proteins.

Molecular characterization of Trichomonas vaginalis isolates from the Philippines.

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Gamma+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.