[Show abstract][Hide abstract] ABSTRACT: Transformation of Chlamydia trachomatis should greatly advance the chlamydial research. However, significant progress has been hindered by the failure of C. trachomatis to induce clinically relevant pathology in animal models. Chlamydia muridarum, which naturally infects mice, can induce hydrosalpinx in mice, a tubal pathology also seen in women infected with C. trachomatis. We have developed a C. muridarum transformation system and confirmed Pgp1, -2, -6, and -8 as plasmid maintenance factors, Pgp3, -5, and -7 as dispensable
for in vitro growth, and Pgp4 as a positive regulator of genes that are dependent on plasmid for expression. More importantly, we have
discovered that Pgp5 can negatively regulate the same plasmid-dependent genes. Deletion of Pgp5 led to a significant increase
in expression of the plasmid-dependent genes, suggesting that Pgp5 can suppress the expression of these genes. Replacement
of pgp5 with a mCherry gene, or premature termination of pgp5 translation, also increased expression of the plasmid-dependent genes, indicating that Pgp5 protein but not its DNA sequence
is required for the inhibitory effect. Replacing C. muridarum pgp5 with a C. trachomatis pgp5 still inhibited the plasmid-dependent gene expression, indicating that the negative regulation of plasmid-dependent genes
is a common feature of all Pgp5 regardless of its origin. Nevertheless, C. muridarum Pgp5 is more potent than C. trachomatis Pgp5 in suppressing gene expression. Thus, we have uncovered a novel function of Pgp5 and developed a C. muridarum transformation system for further mapping chlamydial pathogenic and protective determinants in animal models.
Preview · Article · Dec 2013 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: We previously reported that the Chlamydia trachomatis outer membrane complex protein B (OmcB) was partially processed in Chlamydia-infected cells. We have now confirmed that the OmcB processing occurred inside live cells during chlamydial infection and
was not due to proteolysis during sample harvesting. OmcB processing was preceded by the generation of active CPAF, a serine
protease known to be able to cross the inner membrane via a Sec-dependent pathway, suggesting that active CPAF is available
for processing OmcB in the periplasm. In a cell-free system, CPAF activity is both necessary and sufficient for processing
OmcB. Both depletion of CPAF from Chlamydia-infected cell lysates with a CPAF-specific antibody and blocking CPAF activity with a CPAF-specific inhibitory peptide removed
the OmcB processing ability of the lysates. A highly purified wild-type CPAF but not a catalytic residue-substituted mutant
CPAF was sufficient for processing OmcB. Most importantly, in chlamydial culture, inhibition of CPAF with a specific inhibitory
peptide blocked OmcB processing and reduced the recovery of infectious organisms. Thus, we have identified OmcB as a novel
authentic target for the putative chlamydial virulence factor CPAF, which should facilitate our understanding of the roles
of CPAF in chlamydial biology and pathogenesis.
Full-text · Article · Dec 2012 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.
[Show abstract][Hide abstract] ABSTRACT: The Chlamydia trachomatis outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB,
we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody
detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals
in C. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and
OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were
retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after the C. trachomatis-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also
led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing
and release occurred in all biovars of C. trachomatis. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic in C. trachomatis-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied
by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB
as a target for the development of diagnosis methods and vaccines.
Full-text · Article · Mar 2011 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: The Chlamydia-specific hypothetical protein CT795 was dominantly recognized by human antisera produced during C. trachomatis infection but not by animal antisera raised against dead chlamydia organisms. The immundominant region recognized by the human antibodies was mapped to the N-terminal fragment T22-S69. The endogenous CT795 was detected in the cytoplasm of host cells during C. trachomatis infection and was highly enriched in the host cytosolic fraction but absent in the purified chlamydia organisms, suggesting that CT795 is synthesized and secreted into host cell cytoplasm without incorporation into the organisms. All C. trachomatis serovars tested secreted CT795. A predicted signal peptide of CT795 directed the mature PhoA to cross Escherichia coli inner membranes. The secretion of CT795 in Chlamydia-infected cells was inhibited by a C(16) compound targeting signal peptidase I, but not by a C(1) compound known to block the type III secretion pathway. These results suggest that CT795, like CPAF (a Chlamydia-secreted virulence factor), is secreted into the host cell cytoplasm via a sec-dependent mechanism and not by a type III secretion pathway. The above characterizations of CT795 have provided important information for further understanding the potential roles of CT795 in C. trachomatis pathogenesis.
Full-text · Article · Mar 2011 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Chlamydial interactions with host cells rely on chlamydia-secreted proteins, including preexisting and newly synthesized proteins. The preexisting proteins released from EBs participate in both chlamydial entry and intracellular survival at the early stage while the newly synthesized proteins secreted from RBs can both manipulate host cell signaling pathways and promote chlamydial reinfection. Despite the significant progresses made in the past decade, the precise mechanisms on what and how chlamydia-secreted proteins interact with host cells remain largely unknown, and will therefore still represent major research directions of the chlamydial field in the foreseeable future. Development of more effective methodologies will aid in discovery of new secretion molecules and novel mechanisms utilized in chlamydial interactions with host cells. The continuing accumulation of knowledge on chlamydia-secreted proteins and their mechanism of action may lead to discovery of novel prevention and therapeutic reagents for preventing and controlling chlamydial infection and pathologies.
No preview · Article · Dec 2010 · Current Chemical Biology