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.

The density of negative charge in the cell wall influences two-component signal transduction in Bacillus subtilis.

The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with d-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt(+) wild-type strain under a secretion stress condition (hypersecretion of AmyQ alpha-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P(liaI)) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P(liaI) under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS- and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.

Systemic and mucosal antibody response in experimental Chlamydia pneumoniae infection of mice.

Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.

NMR solution structure and characterization of substrate binding site of the PPIase domain of PrsA protein from Bacillus subtilis.

PrsA is a peptidyl-prolyl isomerase (PPIase) from Bacillus subtilis belonging to the parvulin family of PPIases. It is a membrane bound lipoprotein at the membrane-wall interface, involved in folding of exported proteins. We present the NMR solution structure of the PPIase domain of PrsA, the first from a Gram-positive bacterium. In addition we mapped out the active site with NMR titration experiments. A high degree of conservation with other members of the parvulin family was revealed in the structure and binding site. Interactions with substrate peptides were also characterized by mutated domains revealing that H122 is indispensable for overall correct folding.

Cationic antimicrobial peptides elicit a complex stress response in Bacillus subtilis that involves ECF-type sigma factors and two-component signal transduction systems.

Stress responses of Bacillus subtilis to membrane-active cationic antimicrobial peptides were studied. Global analysis of gene expression by DNA macroarray showed that peptides at a subinhibitory concentration activated numerous genes. A prominent pattern was the activation of two extracytoplasmic function sigma factor regulons, SigW and SigM. Two natural antimicrobial peptides, LL-37 and PG-1, were weak activators of SigW regulon genes, whereas their synthetic analogue poly-L-lysine was clearly a stronger activator of SigW. It was demonstrated for the first time that LL-37 is a strong and specific activator of the YxdJK two-component systems, one of the three highly homologous two-component systems sensing antimicrobial compounds. YxdJK regulates the expression of the YxdLM ABC transporter. The LiaRS (YvqCE) TCS was also strongly activated by LL-37, but its activation is not LL-37 specific, as was demonstrated by its activation with PG-1 and Triton X-100. Other strongly LL-37-induced genes included yrhH and yhcGHI. Taken together, the responses to cationic antimicrobial peptides revealed highly complex regulatory patterns and induction of several signal transduction pathways. The results suggest significant overlap between different stress regulons and interdependence of signal transduction pathways mediating stress responses.

Transcriptome analysis of the secretion stress response of Bacillus subtilis.

Transcription profiling of all protein-encoding genes of Bacillus subtilis was carried out under several secretion stress conditions in the exponential growth phase. Cells that secreted AmyQ alpha-amylase at a high level were stressed only moderately: seven genes were induced, most significantly htrA and htrB, encoding quality control proteases, and yqxL, encoding a putative CorA-type Mg(2+) transporter. These three genes were induced more strongly by severe secretion stress (prsA3 mutant secreting AmyQ), suggesting that their expression responds to protein misfolding. In addition, 17 other genes were induced, including the liaIHGFSR (yvqIHGFEC) operon, csaA and ffh, encoding chaperones involved in the pretranslocational phase of secretion, and genes involved in cell wall synthesis/modification. Severe secretion stress caused downregulation of 23 genes, including the prsA paralogue yacD. Analysis of a cssS knockout mutant indicated that the absence of the CssRS two-component system, and consequently the absence of the HtrA and HtrB proteases, caused secretion stress. The results also suggest that the htrA and htrB genes comprise the CssRS regulon. B. subtilis cells respond to secretion/folding stress by various changes in gene expression, which can be seen as an attempt to combat the stress condition.

Post-translocational folding of secretory proteins in Gram-positive bacteria.

The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells. In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form. They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin. It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates. Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions. Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress. Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.

DNA immunization followed by a viral vector booster in a Chlamydia pneumoniae mouse model.

Vaccination against Chlamydia pneumoniae would be a beneficial strategy for either preventing or controlling infection by this human respiratory pathogen that also causes persistent infections. In the present study, we used recombinant Semliki Forest virus (rSFV) particles for delivering C. pneumoniae antigens major outer membrane protein (MOMP) or outer membrane protein 2 (Omp2) to the mice or applied the prime-boost technique, where mice were first primed with naked DNA and then boosted with the viral vector coding for the same proteins. Partial protection suggested by the reduced number of cultivable bacteria from the lungs of the challenged mice was seen in mice immunized by either method with MOMP expressing constructs. A significant protection was also achieved after DNA/rSFV immunization with Omp2. DNA priming followed by rSFV boosting induced a more prominent IFN-gamma production after challenge at the site of the infection in pulmonary and mediastinal cells.

Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis.

The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall. This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase). In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues. PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain. We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth. Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro. Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope.

Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.

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.

The extracytoplasmic folding factor PrsA is required for protein secretion only in the presence of the cell wall in Bacillus subtilis.

Pulse-chase labelling was used to study the role of the cell wall microenvironment in the functioning of Bacillus subtilis PrsA, an extracellular lipoprotein and member of the parvulin family of peptidylprolyl cis/trans-isomerases. It was found that in protoplasts, and thus in the absence of a cell wall matrix, the post-translocational folding, stability and secretion of the AmyQ alpha-amylase were independent of PrsA, in contrast to the strict dependency found in rods. The results indicate that PrsA is dedicated to assisting the folding and stability of exported proteins in the particular microenvironment of the cytoplasmic membrane-cell wall interface, possibly as a chaperone preventing unproductive interactions with the wall. The data also provide evidence for a crucial role of the wall in protein secretion. The presence of the wall directly or indirectly facilitates the release of AmyQ from the cell membrane and affects the rate of the signal peptide processing.

Identification of Chlamydia pneumoniae-derived mouse CD8 epitopes.

Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8(+) T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8(+) T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8(+) T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8(+) T-cell function in vivo and generation of epitope-specific prevention strategies.

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.