Corrigendum: Chlamydia pneumoniae CdsL regulates CdsN ATPase activity, and disruption with a peptide mimetic prevents bacterial invasion.

[This corrects the article DOI: 10.3389/fmicb.2011.00021.].

Enumeration of Viable Chlamydia from Infected Animals Using Immunofluorescent Microscopy.

An appropriate means of quantitating infectious Chlamydia from infected animals is essential for the evaluation of vaccines. However, unlike methods involving culture, nonculture methods, including detection of antigen or DNA, are not able to differentiate between viable and nonviable organisms. As an obligate intracellular bacterium, Chlamydia replicates inside host cells by forming unique organelles called inclusions. Here, we describe the enumeration of viable C. trachomatis from infected mice by culturing vaginal swabs on McCoy cells and counting inclusions via immunofluorescent microscopy.

Low-cost and versatile integration of microwire electrodes and optical waveguides into silicone elastomeric devices using modified xurographic methods.

Microelectrodes are used in microfluidic devices for a variety of purposes such as heating, applying electric fields, and electrochemical sensing. However, they are still manufactured by expensive deposition techniques such as sputtering or evaporation and patterned using photolithography methods. More recently, alternate methods including nanoparticle sintering and use of liquid metal flowing through microchannels have been used to fabricate microelectrodes. These methods are limited in the material choices or require post processing to be integrated into microchannels. Here we developed a low-cost and versatile method to integrate high-quality metal microwires into polydimethylsiloxane (PDMS) using xurography. The microwire integration process includes cutting slit pattern on PDMS substrate and subsequent writing metal microwires into the slit pattern using a specialized tip. Then the microwire-integrated PDMS was sealed/bonded using uncured PDMS prepolymer. This method enables integration of metal microwires of diameter as small as 15 µm into PDMS devices. Integration of multiple microwires with minimum spacing of 150 µm has also been demonstrated. The versatility of this method is demonstrated by the fabrication of metal microwire suspended in the middle of the microchannel, which is difficult to achieve using conventional electrode fabrication methods. This low-cost method avoids expensive clean room fabrication yet producing high-quality electrodes and can be used in a variety of microfluidic and MEMS applications.

Hospital admissions for lower respiratory tract infections among infants in the Canadian Arctic: a cohort study.

BACKGROUND: It is unknown whether this burden of disease of lower respiratory tract infections is comparable across the Canadian Arctic. The objectives of this surveillance study were to compare the rates of hospital admission for lower respiratory tract infection and the severity of infection across Arctic Canada, and to describe the responsible viruses. METHODS: We performed a prospective multicentre surveillance study of infants less than 1 year of age admitted in 2009 with lower respiratory tract infection to all hospitals (5 regional, 4 tertiary) in the Northwest Territories, Nunavut and Nunavik to assess for regional differences. Nasopharyngeal aspirates were processed by means of a polymerase chain reaction respiratory viral panel, testing for 20 respiratory viruses and influenza A (H1N1). The role of coinfection was assessed by means of regression analysis for length of stay (short: < 7 d; long: > 14 d). Outcomes compared included rates of lower respiratory tract infection, respiratory syncytial virus infection, transfer to tertiary hospital and severe lower respiratory tract infection (respiratory failure, intubation and mechanical ventilation, and/or cardiopulmonary resuscitation). RESULTS: There were 348 admissions for lower respiratory tract infection in the population of interest in 2009. Rates of admission per 1000 live births varied significantly, from 39 in the Northwest Territories to 456 in Nunavik (p < 0.001). The rates of tertiary admissions and severe lower respiratory tract infection per 1000 live births in the Northwest Territories were 5.6 and 1.4, respectively, compared to 55.9 and 17.1, respectively, in Nunavut and 52.0 and 20.0, respectively, in Nunavik (p ≤ 0.001). Respiratory syncytial virus was the most common virus identified (124 cases [41.6% of those tested]), and coinfection was detected in 51 cases (41.1%) of infection with this virus. Longer length of stay was associated with coinfection (odds ratio [OR] 2.64) and underlying risk factors (OR 4.39). Length of stay decreased by 32.2% for every 30-day increase in age (OR 0.68). INTERPRETATION: Nunavut and Nunavik have very elevated rates of lower respiratory tract infection, with severe outcomes. Respiratory syncytial virus was the most common virus identified, and coinfection was associated with longer length of stay. Targeted public health interventions are required to reduce the burden of disease for infants residing in these Arctic regions.

Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge.

Chlamydia trachomatis infections in women are often asymptomatic and if left untreated can lead to significant late sequelae including pelvic inflammatory disease and tubal factor infertility. Vaccine development efforts over the past three decades have been unproductive and there is no vaccine approved for use in humans. The existence of serologically distinct strains or serovars of C. trachomatis mandates a vaccine that will provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of both structural and effector proteins which is an essential virulence factor for infection and intracellular replication. In this study we evaluated a novel fusion protein antigen (BD584) which consists of three T3SS proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydial vaccine candidate. Intranasal immunization with BD584 elicited serum neutralizing antibodies that inhibited C. trachomatis infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding from the vagina at the peak of infection and cleared the infection sooner than control mice. Immunization with BD584 also reduced the rate of hydrosalpinx by 87.5% compared to control mice. Together, these results suggest that highly conserved proteins of the chlamydial T3SS may represent good candidates for a Chlamydia vaccine.

Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

BACKGROUND: Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS. RESULTS: In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells. CONCLUSION: The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.

Targeting PI3K-p110α Suppresses Influenza Virus Infection in Chronic Obstructive Pulmonary Disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD) and influenza virus infections are major global health issues. Patients with COPD are more susceptible to infection, which exacerbates their condition and increases morbidity and mortality. The mechanisms of increased susceptibility remain poorly understood, and current preventions and treatments have substantial limitations. OBJECTIVES: To characterize the mechanisms of increased susceptibility to influenza virus infection in COPD and the potential for therapeutic targeting. METHODS: We used a combination of primary bronchial epithelial cells (pBECs) from COPD and healthy control subjects, a mouse model of cigarette smoke-induced experimental COPD, and influenza infection. The role of the phosphoinositide-3-kinase (PI3K) pathway was characterized using molecular methods, and its potential for targeting assessed using inhibitors. MEASUREMENTS AND MAIN RESULTS: COPD pBECs were susceptible to increased viral entry and replication. Infected mice with experimental COPD also had more severe infection (increased viral titer and pulmonary inflammation, and compromised lung function). These processes were associated with impaired antiviral immunity, reduced retinoic acid-inducible gene-I, and IFN/cytokine and chemokine responses. Increased PI3K-p110α levels and activity in COPD pBECs and/or mice were responsible for increased infection and reduced antiviral responses. Global PI3K, specific therapeutic p110α inhibitors, or exogenous IFN-ß restored protective antiviral responses, suppressed infection, and improved lung function. CONCLUSIONS: The increased susceptibility of individuals with COPD to influenza likely results from impaired antiviral responses, which are mediated by increased PI3K-p110α activity. This pathway may be targeted therapeutically in COPD, or in healthy individuals, during seasonal or pandemic outbreaks to prevent and/or treat influenza.

Chlamydia pneumoniae CopD translocator protein plays a critical role in type III secretion (T3S) and infection.

Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120-125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.

Structural characterization of a novel Chlamydia pneumoniae type III secretion-associated protein, Cpn0803.

Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.

Molecular diagnosis of respiratory virus infections.

The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.

Prognosis of West Nile virus associated acute flaccid paralysis: a case series.

INTRODUCTION: Little is known about the long-term health related quality of life outcomes in patients with West Nile virus associated acute flaccid paralysis. We describe the quality of life scores of seven patients with acute flaccid paralysis who presented to hospital between 2003 and 2006, and were followed for up to two years. CASE PRESENTATIONS: Between 2003 and 2006, 157 symptomatic patients with West Nile virus were enrolled in a longitudinal cohort study of West Nile virus in Canada. Seven patients (4%) had acute flaccid paralysis. The first patient was a 55-year-old man who presented with left upper extremity weakness. The second patient was a 54-year-old man who presented with bilateral upper extremity weakness. The third patient was a 66-year-old woman who developed bilateral upper and lower extremity weakness. The fourth patient was a 67-year-old man who presented with right lower extremity weakness. The fifth patient was a 60-year-old woman who developed bilateral lower extremity weakness. The sixth patient was a 71-year-old man with a history of Parkinson's disease and acute onset bilateral lower extremity weakness. The seventh patient was a 52-year-old man who presented with right lower extremity weakness. All were Caucasian. Patients were followed for a mean of 1.1 years. At the end of follow-up the mean score on the Physical Component Summary of the Short-Form 36 scale had only slightly increased to 39. In contrast, mean score on the Mental Component Summary of the Short-Form 36 scale at the end of follow-up had normalized to 50. CONCLUSION: Despite the poor physical prognosis for patients with acute flaccid paralysis, the mental health outcomes are generally favorable.

Chlamydia Pneumoniae CdsL Regulates CdsN ATPase Activity, and Disruption with a Peptide Mimetic Prevents Bacterial Invasion.

Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S) to invade cells and replicate intracellularly within a cytoplasmic vacuole called an inclusion body. Chlamydia pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN). In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using Pepscan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN(221-229) and CdsN(265-270). We confirmed the binding domains using a pull-down assay and showed that GST-CdsN(221-270), which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from Escherichia coli, EscN, to map the binding domains on the predicted 3D structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL-CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and chlamydial invasion. Treatment of elementary body with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses.

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.

Expectant management versus immediate treatment for low-grade cervical intraepithelial neoplasia : a randomized trial in Canada and Brazil.

BACKGROUND: The optimal management strategy for women with low-grade biopsy-proven cervical intraepithelial neoplasia (CIN) is not clear. Our objective was to compare the effectiveness of regular colposcopic follow-up and treatment of progressive disease only versus immediate treatment. METHODS: Data were accrued between November 2000 and March 2006 for a noninferiority randomized clinical trial of 415 women with biopsy-proven grade 1 CIN from 8 Canadian and 2 Brazilian colposcopy clinics. Subjects were randomly assigned to either undergo immediate treatment with a loop electrical excision procedure (LEEP) or receive regular colposcopic follow-up for 18 months. The primary outcome was progression of disease to CIN 2 to 3 was based on histology obtained during 18 months of follow-up. Treatments were compared using differences of proportion with a 9% noninferiority margin. Analysis was conducted on the basis of intention-to-treat. RESULTS: An initial LEEP was performed on 179 women. Disease progression was found in 32. Easily controlled vaginal bleeding occurred in 16 (8.9%). During follow-up, disease progression was identified in 3 (1.7%) women in the immediate treatment arm and 9 (4.4%) in the colposcopic follow-up arm-a tolerable difference of 2.7% with 1-sided 95% confidence interval (CI) upper limit of 6.0%. Compliance with all 3 follow-up visits was 61% overall, but significantly worse in women ≤30 years of age (P < .05). CONCLUSIONS: The risk of progression to CIN grade 2 or 3 or cancer over 18 months was similar in the 2 treatment groups. In Canada and Brazil, follow-up for 18 months is a reasonable management strategy for women with persistent low-grade cytology who are found to have grade 1 CIN on referral for colposcopy and cervical biopsy.

Nucleic acid amplification-based diagnosis of respiratory virus infections.

The appearance of eight new respiratory viruses in the human population in the past 9 years, including two new pandemics (SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009), has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid amplification tests (NATs) that first appeared two decades ago have been developed for both conventional and emerging viruses and now form the backbone of the clinical laboratory. NATs provide fast, accurate and sensitive detection of respiratory viruses and have significantly increased our understanding of the epidemiology of these viruses. Multiplex PCR assays have been introduced recently and several commercial tests are now available. The final chapter in the evolution of respiratory virus diagnostics will be the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance to multiplex assays. These resistance assays together with new viral load tests will enable clinical laboratories to provide physicians with important information for optimal treatment of patients.

Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.

BACKGROUND: The identification of influenza A virus subtypes in clinical specimens is becoming increasingly important for clinical laboratories since seasonal H1N1, H3N2 and pandemic H1N1 influenza A viruses can have defined antiviral resistance patterns and subtyping can be used as a surrogate for antiviral resistance testing. OBJECTIVES: To develop a novel multiplex PCR (M-PCR) assay for the combined identification of influenza A subtype and oseltamivir resistance (H275Y) genotype in a combined assay format using Luminex xMAP™ technology. STUDY DESIGN: The M-PCR assay employed five degenerate primers to amplify the hemagglutinin (HA) and neuraminidase (NA) genes and eight tagged primers in a target specific primer extension reaction (TSPE). Products were analysed using xTAG™ beads containing specific anti-tag oligonucleotides. RESULTS: M-PCR correctly identified the subtype for 54/54 specimens that were influenza A positive, including 13/13 seasonal H3N2, 17/17 seasonal H1N1 and 24/24 pandemic H1N1 for both HA and NA genes. For oseltamivir resistance the M-PCR assay correctly identified 41/41 H1N1 viruses as oseltamivir sensitive (H275) or resistant (H275Y). Analysis of sequential specimens from two immunocompromised patients revealed the appearance of the H275Y allele at earlier time points after infection compared with Sanger sequencing. CONCLUSIONS: The combined M-PCR assay correctly subtyped seasonal and pandemic influenza A viruses and accurately detected the H275Y oseltamivir resistance allele. This assay should provide useful information to clinicians for appropriate patient management.

Development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing.

We developed and evaluated flocked nasal midturbinate swabs obtained from 55 asymptomatic and 108 symptomatic volunteers. Self-collected swabs obtained from asymptomatic volunteers yielded numbers of respiratory epithelial cells comparable to those of staff-collected nasal (n = 55) or nasopharyngeal (n = 20) swabs. Specific viruses were detected in swabs self-collected by 42/108 (38.9%) symptomatic volunteers by multiplex PCR.

The role of cytology (Pap tests) and human papillomavirus testing in anal cancer screening.

OBJECTIVE: To assess anal oncogenic human papillomavirus (HPV) and anal cytology as screening tests for detecting high-grade anal intraepithelial neoplasia (AIN 2+), as this is an immediate anal cancer precursor. DESIGN: Cross-sectional study of 401 HIV-positive men who have sex with men (MSM). The endpoint was histologically confirmed AIN 2+ obtained by high-resolution anoscopy. Cytology and biopsy specimens were assigned random numbers and independently assessed by two pathologists. METHODS: We did concomitant anal cytology, anal HPV testing and HRA with directed biopsies without knowing the results of each intervention. The main outcome measures were the sensitivity, specificity, negative predictive value and positive predictive value of anal cytology and oncogenic HPV for the detection of AIN 2+. RESULTS: Cytology was abnormal in 67% of patients: high-grade squamous intraepithelial lesion, 12%; low-grade squamous intraepithelial lesion, 43% and atypical squamous cells of undetermined significance, 12%. Biopsies were abnormal in 68% of patients: AIN 2+, 25% and AIN 1, 43%. HPV was detected in 93% with multiple HPV types in 92% and oncogenic HPV types in 88%. Test performance characteristics for the detection of AIN 2+ using any abnormality on anal cytology were: sensitivity 84%, specificity 39%, negative predictive value 88% and positive predictive value 31%; using oncogenic HPV: sensitivity 100%, specificity 16%, negative predictive value 100% and positive predictive value 28%. CONCLUSION: Anal cytology and HPV detection have high sensitivity but low specificity for detecting AIN 2+. HIV-positive men who have sex with men have a high prevalence of AIN 2+ and require high-resolution anoscopy for optimal detection of high-grade anal dysplasia.

Interactions between flagellar and type III secretion proteins in Chlamydia pneumoniae.

BACKGROUND: Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown. RESULTS: Full length FliI, and fragments of FlhA, FliF, and FliI, were cloned and expressed as either GST or His tagged proteins in E. coli. The GST-tagged full length FliI protein was shown to possess ATPase activity, hydrolyzing ATP at a rate of 0.15 +/- .02 micromol min-1 mg-1 in a time- and dose-dependant manner. Using bacterial-2-hybrid and GST pull-down assays, the N-terminal domain of FliI was shown to interact with the cytoplasmic domain of FlhA, but not with FliF, and the cytoplasmic domain of FlhA was shown to interact with the C-terminus of FliF. The absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type III secretion proteins, and we found that FliI interacted with CdsL and CopN, while FlhA interacted with CdsL and Cpn0322 (YscU ortholog CdsU). CONCLUSIONS: The specific interaction of the four orthologous flagellar proteins in C. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with T3S components, suggests a role in bacterial replication and/or intracellular survival.

A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication.

BACKGROUND: We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. RESULTS: Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. CONCLUSION: We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.