Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues

Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.

Induction of partial immunity in both males and females is sufficient to protect females against sexual transmission of Chlamydia

Sexually transmitted Chlamydia trachomatis causes infertility, and because almost 90% of infections are asymptomatic, a vaccine is required for its eradication. Mathematical modeling studies have indicated that a vaccine eliciting partial protection (non-sterilizing) may prevent Chlamydia infection transmission, if administered to both sexes before an infection. However, reducing chlamydial inoculum transmitted by males and increasing infection resistance in females through vaccination to elicit sterilizing immunity has yet to be investigated experimentally. Here we show that a partially protective vaccine (chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) provided sterilizing immunity against sexual transmission between immunized mice. Immunizing male or female mice before an infection reduced chlamydial burden and disease development, but did not prevent infection. However, infection and inflammatory disease responsible for infertility were absent in 100% of immunized female mice challenged intravaginally with ejaculate collected from infected immunized males. In contrast to the sterilizing immunity generated following recovery from a previous chlamydial infection, protective immunity conferred by MOMP/IMX occurred independent of resident memory T cells. Our results demonstrate that vaccination of males or females can further protect the opposing sex, whereas vaccination of both sexes can synergize to elicit sterilizing immunity against Chlamydia sexual transmission.

Human and pathogen factors associated with chlamydia trachomatis-related infertility in women

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen worldwide. Infection can result in serious reproductive pathologies, including pelvic inflammatory disease, ectopic pregnancy, and infertility, in women. However, the processes that result in these reproductive pathologies have not been well defined. Here we review the evidence for the human disease burden of these chlamydial reproductive pathologies. We then review human-based evidence that links Chlamydia with reproductive pathologies in women. We present data supporting the idea that host, immunological, epidemiological, and pathogen factors may all contribute to the development of infertility. Specifically, we review the existing evidence that host and pathogen genotypes, host hormone status, age of sexual debut, sexual behavior, coinfections, and repeat infections are all likely to be contributory factors in development of infertility. Pathogen factors such as infectious burden, treatment failure, and tissue tropisms or ascension capacity are also potential contributory factors. We present four possible processes of pathology development and how these processes are supported by the published data. We highlight the limitations of the evidence and propose future studies that could improve our understanding of how chlamydial infertility in women occurs and possible future interventions to reduce this disease burden.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent

Background The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. Results In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. Conclusions Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.

Major histocompatibility complex and coronary artery disease: Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells.

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an "inclusion". Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.

Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.

Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.

Pilot study of the utility and acceptability of tampon sampling for the diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infections by duplex realtime polymerase chain reaction in United Kingdom sex workers

We aimed to evaluate the acceptability of self-collected tampon samples for the screening of female sex workers for sexually transmitted infections. We recruited 65 sex workers, and 63 agreed to provide tampon samples. The tampon samples were processed by realtime polymerase chain reaction (PCR) targeting Neisseria gonorrhoeae and Chlamydia trachomatis. Urethral and endocervical swabs were also obtained from 61 of 63 participants and tested using culture (N. gonorrhoeae) and the BD ProbeTec strand displacement amplification (SDA) (C. trachomatis) assay. Tampon sampling was preferred by 95% of the women and all favoured being tested away from genitourinary medicine clinics; the most common reasons cited were avoidance of embarrassment (40%) and convenience (30%). Besides near-universal acceptability of tampon sampling, the tampon sampling-PCR approach described in this study appeared to have enhanced sensitivity compared with conventional testing, suggesting the possibility of a residual hidden burden of N. gonorrhoeae and/or C. trachomatis genital infections in UK female sex workers.

Chlamydia trachomatis Infecção na grávida e no recém-nascido

A infecção urogenital causada por Chlamydia trachomatis é a doença bacteriana sexualmente transmissível mais comum na Europa, tanto em homens como em mulheres. Constitui um grave problema de saúde pública devido à elevada percentagem de portadores assintomáticos, às complicações clínicas que daí podem resultar e à possibilidade de transmissão vertical. O presente trabalho teve como principais objectivos: i) avaliar a prevalência da infecção por C. trachomatis e por Neisseria gonorrhoeae num grupo de grávidas de 36 semanas atendidas na consulta externa de Obstetrícia do Hospital Amadora Sintra e nos recém-nascidos de mães infectadas, ii) identificar os serovares responsáveis pelas infecções por C. trachomatis, iii) verificar a distribuição da prevalência da infecção por C. trachomatis em função da idade e iv) avaliar a utilidade de uma técnica de PCR multiplex e de PCR multiplex em tempo real no diagnóstico desta infecção. Foram testadas 1201 amostras de urina do primeiro jacto de grávidas e 18 exsudados oculares provenientes de recém-nascidos cujas mães estavam infectadas com C. trachomatis. Cada amostra foi testada pelas técnicas de PCR multiplex e de PCR multiplex em tempo real, tendo como alvos de amplificação um fragmento do plasmídio críptico e outro do gene omp1. Todos os resultados positivos foram confirmados com uma técnica de nested PCR e posteriormente enviados para sequenciação para identificação dos serovares envolvidos. Em todas as amostras foi ainda pesquisada a presença de ADN de N. gonorrhoeae através de técnicas de PCR e de PCR em tempo real sendo que, na primeira, o alvo a amplificar foi um fragmento do gene ccpB do plasmídio pJDI e na segunda o pseudogene porA. Os resultados positivos foram confirmados por RFLP. A prevalência da infecção por C. trachomatis e por N. gonorrhoeae foi de 3,7% (45/1201) e de 0,08% (1/1201), respectivamente. Nos recém-nascidos, a prevalência foi de 0% para ambas as infecções, embora o número de recém-nascidos estudados (18/45) dificilmente seja representativo. O serovar mais prevalente foi o E (31,1%), seguido do G (15,6%), do D/Da (13,3%), do F, I/Ia e do J (11,1%). O serovar K foi identificado em 4,4% das amostras infectadas e o H em apenas 2,2%. A técnica de PCR multiplex em tempo real parece ser mais adequada para o diagnóstico da infecção por C. trachomatis do que a técnica de PCR multiplex, tendo a primeira detectado 100% dos casos de infecção por este microrganismo (45/45), enquanto que a segunda detectou apenas 71% (32/45) dos mesmos.