Pseudomonas aeruginosa is a prevalent opportunistic pathogen that causes fatal infections in immunocompromised individuals. Quorum sensing (QS) is a cell-to-cell communication process that controls virulence gene expression and biofilm formation in P. aeruginosa. Here, the QS systems and the relevant virulence traits in clinical P. aeruginosa isolates were characterized. Eleven out of the ninety-four P. aeruginosa isolates exhibited a biofilm-deficient phenotype. Two biofilm-deficient isolates, one from blood and the one from pleural effusion, appeared to carry a same mutation in lasR. These two isolates differed in the ability to produce QS-regulated virulence factors, but contained the same functionally deficient LasR with the truncated C-terminal domains and belonged to the same multilocus sequence type (ST227). Chromosomal lasR complementation in these lasR mutants verified that lasR inactivation was the sole cause of las deficiency. LasR was not absolutely required for rhl signal in these lasR mutants, suggesting the presence of lasR-independent QS systems. We provided evidence that the virulence gene expression are not regulated in the same manner in these isolates. These results support the hypothesis that conventional QS hierarchy can be smashed by naturally occurring lasR mutation in clinical P. aeruginosa isolates and that complex QS hierarchy may play a role in maintaining infection of this opportunistic pathogen.

The obligate intracellular human pathogen Chlamydia trachomatis undergoes a complex developmental program involving transition between two forms: the infectious elementary body (EB), and the rapidly dividing reticulate body (RB). However, the regulators controlling this development have not been identified. To uncover potential regulators of transcription in C. trachomatis, we screened a C. trachomatis genomic library for sequences encoding proteins that interact with RNA polymerase (RNAP). We report the identification of one such protein, CT663, which interacts with the β and σ subunits of RNAP. Specifically, we show that CT663 interacts with the flap domain of the β subunit (β-flap) and conserved region 4 of the primary σ subunit (σ⁶⁶ in C. trachomatis). We find that CT663 inhibits σ⁶⁶-dependent (but not σ²⁸-dependent) transcription in vitro, and we present evidence that CT663 exerts this effect as a component of the RNAP holoenzyme. The analysis of C. trachomatis-infected cells reveals that CT663 begins to accumulate at the commencement of the RB-to-EB transition. Our findings suggest that CT663 functions as a negative regulator of σ⁶⁶-dependent transcription, facilitating a global change in gene expression. The strategy used here is generally applicable in cases where genetic tools are unavailable.

Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818–1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.

We used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.