A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.

Soroepidemiologia de Chlamydia trachomatis, Chlamydia pneumoniae e Treponema pallidum em portadores do Vírus da imunodeficiência humana (HIV), no Estado do Pará

A Chlamydia trachomatis e o Treponema pallidum compartilham com o HIV uma importante forma de transmissão: a via sexual. Por conta do comprometimento imunológico dos portadores de HIV, a C. pneumoniae pode apresentar um papel potencial em infecções respiratórias. Este trabalho objetivou a descrição da soroprevalência destes três agentes em portadores de HIV do Estado do Pará, Brasil. Entre setembro de 2007 a junho de 2008, foram coletadas 430 amostras de portadores de HIV em Belém, Pará. Estas foram submetidas a um ELISA para detecção de anticorpo IgG e IgM anti-Chlamydia e, dentre os positivos, uma amostragem aleatória foi escolhida e submetida à microimunofluorescência para sorotipagem. Para a detecção de anticorpos anti-Treponema pallidum foi feito um teste não treponêmico (RPR) e um teste treponêmico (ELISA). Os resultados obtidos foram analisados pelo teste do χ². A prevalência geral de anticorpos anti-Chlamydia foi 64,2% (51,6% para IgG e 4% para IgM). A sorotipagem mostrou uma alta prevalência de C. trachomatis (100% tanto para IgG como IgM), e C. pneumoniae (73,5% IgG e 70,5% IgM), sendo que houve uma larga disseminação dos sorotipos que causam infecções genitais da Chlamydia trachomatis. A prevalência geral de anticorpos contra o Treponema pallidum foi de 34,9%, sendo que 7,3% apresentaram resultado laboratorial indicativo de sífilis. As variáveis que apresentaram associação com a infecção por Chlamydia e Treponema pallidum foram: o gênero masculino, maior idade, baixa escolaridade, número de parceiros por semana, a prática de sexo anal, homossexualismo/bissexualismo, uso de droga não-endovenosa, histórico de IST. Faz-se necessário tanto a conscientização como o monitoramento da população, para impedir a transmissão destes agentes e para a melhoria da qualidade de vida dos indivíduos portadores de HIV.

Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10% of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma

Background Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

Ecoepidemiología de Chlamydophila psittaci, pecorum, pneumoniae, abortus en Córdoba

Las Clamidias son bacterias patógenas de los animales de producción, de vida silvestre y de compañía. Además de las pérdidas económicas que producen las infecciones en los planteles de producción bovina, ovina, caprina, porcina y aves de corral, la mayoría de las especies tienen importancia zoonótica, pudiendo dar origen a infecciones graves, potencialmente letales en el ser humano. El orden Chlamydiales está integrado por bacterias que actúan como parásitos intracelulares obligados que desarrollan su ciclo de vida únicamente dentro de inclusiones citoplasmáticas. En este orden se encuentra la familia Chlamydiaceae que comprende dos géneros, Chlamydia y Chlamydophila; y las especies, Chlamydia trachomatis, C. suis, C. muridarum, Chlamydophila psittaci, C. abortus, C. felis, C. caviae, C. pecorum, y C. pneumoniae. C. psittaci causa psitacosis o clamidiosis aviar. En Argentina, los primeros casos clínicos de psitacosis fueron reportados en 1929. Los criadores de aves y quienes las poseen como mascotas, representan el grupo de mayor riesgo; pero también las personas que trabajan en pajarerías y aquellas que por su empleo se ven expuestas a contraer la enfermedad (empleados en peladeros donde se carnean y procesan pollos y otras aves para consumo, veterinarios, empleados de zoológicos, etc.). La infección en humanos se presenta como una neumonía severa; con fiebre alta, escalofríos, dolor de cabeza, mialgia y dificultad respiratoria. Ocasionalmente puede presentarse vómitos, dolor abdominal, diarrea y complicaciones como miocarditis, endocarditis, encefalitis, ictericia y fallas multiorgánicas, que pueden ser fatales sino se le administra el tratamiento adecuado. La infección en las mujeres embarazadas puede producir neumonía, hepatitis, insuficiencia renal, sepsis, parto prematuro y muerte fetal. Existen más de 465 especies de aves en las que se registró C. psittaci, incluyendo ornamentales, de corral, silvestres, acuáticas y palomas. Las patologías que pueden producir en estos animales son neumonitis, conjuntivitis, encefalomielitis, placentopatías, fetopatías, anorexia, diarrea e infecciones persistentes asintomáticas u oligosintomáticas. En bovinos, C. pecorum, C. abortus y C. psittaci producen infecciones respiratorias y genitales; que se presentan como cuadros de enteritis, artritis, encefalomielitis, endometritis e hipofertilidad. En Argentina, la infección clamidial en el ganado caprino fue asociada a daños en el tejido uterino, abortos, partos prematuros y crías débiles. En equinos, C. psittaci y C. pneumoniae producen abortos y desórdenes respiratorios, con un gran impacto en ganadería que redunda en pérdidas económicas. Considerando que existen escasos estudios eco-epidemiológicos y clínicos que reporten el estado de situación de estas infecciones en nuestro medio, es que el presente trabajo propone actualizar y profundizar el conocimiento de las especies de Clamidias de importancia médico-veterinaria presentes en la provincia de Córdoba, Argentina. El desarrollo de este proyecto aportará la implementación de técnicas que mejorarán el diagnóstico microbiológico, confirmarán los cuadros clínicos; y por lo tanto contribuirá al conocimiento de estos agentes infecciosos en nuestra región. Esta información es indispensable para los organismos responsables de la Salud Pública (Ministerios de Salud y Educación, Municipios, etc.) para que puedan obrar en consecuencia y generar sistemas de alerta temprana, tomar medidas de prevención y medidas de control frente a la presencia de un brote epidémico por alguna cepa clamidial.

Mutation frequency of non-ESBL phenotype SENTRY (Asia-Pacific) isolates of Klebsiella pneumoniae conversion to an ESBL positive phenotype

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

Evidence that human chlamydia pneumoniae was zoonotically acquired

Zoonotic infections are a growing threat to global health. Chlamydia pneumoniae is a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease. C. pneumoniae was first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species of Chlamydia, C. pecorum and C. pneumoniae. Transmission of C. pneumoniae between animals and humans has not been reported; however, two other chlamydial species, C. psittaci and C. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain of C. pneumoniae (LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates of C. pneumoniae show that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) of C. pneumoniae which adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Chlamydia pneumoniae is genetically diverse in animals and appears to have crossed the host barrier to humans on (at least) two occasions

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non- Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.

Comparison of koala LPCoLN and human strains of Chlamydia pneumoniae highlights extended genetic diversity in the species

Background Chlamydia pneumoniae is a widespread pathogen causing upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. Previous whole genome analyses have focused on four essentially clonal (> 99% identity) C. pneumoniae human genomes (AR39, CWL029, J138 and TW183), providing relatively little insight into strain diversity and evolution of this species. Results We performed individual gene-by-gene comparisons of the recently sequenced C. pneumoniae koala genome and four C. pneumoniae human genomes to identify species-specific genes, and more importantly, to gain an insight into the genetic diversity and evolution of the species. We selected genes dispersed throughout the chromosome, representing genes that were specific to C. pneumoniae, genes with a demonstrated role in chlamydial biology and/or pathogenicity (n = 49), genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6), and extrachromosomal elements (9 plasmid and 2 bacteriophage genes). Conclusions We have identified strain-specific differences and targets for detection of C. pneumoniae isolates from both human and animal origin. Such characterisation is necessary for an improved understanding of disease transmission and intervention.