Chlamydia pneumoniae entry into epithelial cells by clathrin-independent endocytosis.

A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MßCD) and cholesterol-loading MßCD complexed cholesterol (chol-MßCD). The invasion was attenuated by MßCD-treatment while chol-MßCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells.

Production of Chlamydia pneumoniae proteins in Bacillus subtilis and their use in characterizing immune responses in the experimental infection model.

Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.

ClpXP protease regulates the signal peptide cleavage of secretory preproteins in Bacillus subtilis with a mechanism distinct from that of the Ecs ABC transporter.

Identification and characterization of a suppressor mutation, sup-15, which partially restored secretion in the protein secretion-deficient Bacillus subtilis ecsA26 mutant, led us to discover a novel function of Clp protease. Inactivation of ClpP improved the processing of the precursor of AmyQ alpha-amylase exposed on the outer surface of the cytoplasmic membrane. A similar improvement of AmyQ secretion was conferred by inactivation of the ClpX substrate-binding component of the ClpXP complex. In the absence of ClpXP, the transcription of the sipS, sipT, sipV, and lsp signal peptidase genes was elevated two- to fivefold, a likely cause of the improvement of the processing and secretion of AmyQ and complementation of ecs mutations. Specific overproduction of SipT enhanced the secretion. These findings extend the regulatory roles of ClpXP to protein secretion. ClpXP also influenced the processing of the lipoprotein PrsA. A concerted regulation of signal peptidase genes by a ClpXP-dependent activator is suggested. In contrast, Ecs did not affect transcription of the sip genes, pointing to a different mechanism of secretion regulation.

Molecular analysis of an operon in Bacillus subtilis encoding a novel ABC transporter with a role in exoprotein production, sporulation and competence.

The levels of exoamylase and other exoenzymes of Bacillus subtilis are pleiotropically decreased by the ecs-26 (prs-26) and ecs-13 (prs-13) mutations. These mutations also cause a competence- and sporulation-deficient phenotype. In the present work, the ecs locus, which has been defined by the ecs-26 and ecs-13 mutations, was cloned and sequenced. Sequence analysis revealed a putative operon of three ORFs (ecsA, ecsB and ecsC). ecsA can encode a putative polypeptide of 248 amino acid residues containing an ATP-binding site. The polypeptide shows about 30% sequence similarity with the ATP-binding components of numerous membrane transporters of the ABC-type (ATP-binding cassette transporters or traffic ATPases). The ecs-26 mutation was found to result from a transition of one base pair chaning the glycine164 of EcsA to a glutamic acid residue in the vicinity of the putative ATP-binding pocket. ecsB was predicted to encode a hydrophobic protein with six membrane-spanning helices in a pattern found in other hydrophobic components of ABC transporters. The properties deduced for the ecsA and ecsB gene products are consistent with the interpretation that ecs encodes a novel ABC-type membrane transporter of B. subtilis. The third ORF, ecsC, can encode a putative polypeptide of 237 amino acid residues. The polypeptide does not resemble components of ABC transporters.