Background: Chlamydia is a common bacterial pathogen responsible for many diseases. Methods for transforming this important organism using a β-lactamase as a selection marker have been developed very recently. However, the National Institutes of Health Guidelines for Research Involving Recombinant DNA Molecules do not permit transformation experiments with β-lactamase gene-containing vectors for certain human chlamydial pathogens. Therefore, a different selection marker is urgently needed for transformation of those chlamydiae. Results: After transformation of plasmid-free Chlamydia trachomatis with pGFP:SW2, which carries a β-lactamase and a chloramphenicol acetyltransferase gene fused to a green fluorescence protein gene, transformants were obtained by selection with either ampicillin or chloramphenicol. Stable chloramphenicol-resistant, but ampicillin-sensitive, transformants were obtained using a pGFP:SW2 derivative without the β-lactamase. All transformants expressed green fluorescence protein and had glycogen synthesis activity restored. Conclusions: Chloramphenicol resistance may be used as a selection marker for genetic experiments in Chlamydia. This eliminates the requirement for the use of β-lactamase, of which dissemination to some C. trachomatis serovars may jeopardize clinical treatment of chlamydial infections in pregnant women. Chloramphenicol acetyltransferase may also serve as a useful secondary selection marker for genetic analyses in β-lactamase-transformed chlamydial strains.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear.

Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C . trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

The obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, σ⁶⁶, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor σ²⁸. In prior work, we identified GrgA as a Chlamydia-specific transcription factor that activates σ⁶⁶-dependent transcription by binding DNA and interacting with a non-conserved region (NCR) of σ⁶⁶. Here, we extend these findings by showing GrgA can also activate σ²⁸-dependent transcription through direct interaction with σ²⁸. We measure the binding affinity of GrgA for both σ⁶⁶ and σ²⁸, and we identify regions of GrgA important for σ²⁸-dependent transcription. Similar to results obtained with σ⁶⁶, we find that GrgA's interaction with σ²⁸ involves a NCR located upstream of conserved region 2 of σ²⁸. Our findings suggest GrgA is an important regulator of both σ⁶⁶- and σ²⁸-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.

The obligate intracellular bacteriumChlamydiais a widespread human pathogen that causes serious problems, including (but not limited to) infertility and blindness. Our search for novel antichlamydial metabolites from marine-derived microorganisms led to the isolation of pyocyanin, a small compound fromPseudomonas aeruginosa. Pyocyanin is an effective antichlamydial for all threeChlamydiaspp. tested. It has a 50% inhibitory concentration (IC₅₀) of 0.019 to 0.028 μM, which is comparable to the IC₅₀of tetracycline. At concentrations as low as 0.0039 μM, pyocyanin disables infectivity of the chlamydial elementary body (EB). At 0.5 μM or higher concentrations, the continuous presence of pyocyanin also inhibits chlamydial growth in the inclusion during later stages of the developmental cycle. Oxidative stress, a major known antimicrobial mechanism of pyocyanin, appears to be responsible only for the inhibition of bacterial growth and not for the disinfection of EBs. Pyocyanin is well-tolerated by probiotic vaginalLactobacillusspp. Our findings suggest that pyocyanin is of therapeutic value for chlamydial infections and can serve as a valuable chemical probe for studying chlamydial biology.