Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to InvasiveChlamydia trachomatisBiovars

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachomawhich are distinguished biologically by their natural host cell infection tropism.LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculourogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmiddependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature. IMPORTANCEThe Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes

“…Bacterial plasmids that control chromosomally-encoded genes have been reported 46,47 . However, the broad impact on bacterial genome expression and pathobiology by pAB5 has not been reported for any other plasmid.…”

The Nature and Extent of Plasmid Variation in Chlamydia trachomatis

“…However, CDS6 is nonessential for stable plasmid maintenance in tissue culture [46], and its necessity in vivo has not yet been assessed. Nonetheless, CDS6 encodes the pgp4 protein, which has a role in the plasmid's ability to accumulate glycogen [46] and is the sole regulator of pgp3 [19,86] and other virulence associated genes, suggesting CDS6 is important in the context of the natural host. Along with the presence of only 6 variable sites throughout the gene and the relative rarity of these SNPs in the dataset, this suggests that selective pressure exerted by diagnostic detection will be overcome by natural selection, resulting in its continued low variability.…”

The multiple functions of the numerous Chlamydia trachomatis secreted proteins: the tip of the iceberg

“…The C. trachomatis genes ct142, ct143 , and ct144 genes are organized in an operon encoding three T3S substrates [227, 294]. Strikingly, by IF microscopy, CT142, CT143, and CT144 appear in globular structures outside of chlamydiae resembling those seen with anti-Pls1 and anti-Pls2 antibodies [293–295] (Figure 5A) . Furthermore, CT142, CT143, and CT144 co-localize with each other within the inclusion lumen, suggesting that they could be part of protein complexes [294].…”

“…In summary, Pls1, Pls2, CT142, CT143, and CT144 have unknown function but they are secreted into the lumen of the inclusion where they appear as globular structures, as detected by IF microscopy (Figure 5A) . Coincidently, the genes encoding Pls1, Pls2, CT142, CT143, CT144, and also the glycogen synthase GlgA (see above), are amongst the C. trachomatis chromosomal genes more clearly upregulated by the Chlamydia virulence plasmid through pGP4 [287, 295].…”

“…These effectors mediate fusion between inclusions (IncA), continue subverting host cell vesicular trafficking (IncA, and perhaps CteG and DUF582-containing CT619, CT620, CT621, CT711, and CT712), modulate microtubules (IPAM) and mediate their modification (InaC), promote the assembly of F-actin (InaC) and the redistribution of the Golgi around the inclusion (InaC, Cdu1 and Cdu2), and possibly promote the acquisition of LDs by the inclusion (Lda1, Lda2, and Lda3), interfere with host cell transcription (NUE), and modulate host cell death (CpoS and Cdu1). Finally, chlamydial host cell exit is also controlled by Incs (MrcA and CT228), and by the Chlamydia virulence plasmid, likely through the regulation of expression of T3S effectors and/or of CPAF [251, 295]. Even after chlamydial exit from infected cells, some C. trachomatis effectors concomitantly released from the inclusion lumen and/or host cell cytosol continue to function extracellularly, as is the case of evasion of the innate immune response by CPAF, and possibly by pGP3.…”