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Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

ABSTRACT: Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

SUBMITTER: Heijne M

PROVIDER: S-EPMC8363750 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chlamydia psittaci is the etiological agent of chlamydiosis in birds and can be transmitted to humans, causing severe systemic disease. C. psittaci infects a broad range of hosts; strains are isolated not only from birds but also from mammals, where they seem to have a reduced infectious and zoonotic potential. Comparative analysis of chlamydial genomes revealed the coding sequences of polymorphic membrane proteins (Pmps) to be highly variable regions. Pmps are characterized as adhesins in C. trachomatis and C. pneumoniae and are immunoreactive proteins in several Chlamydia species. Thus, Pmps are considered to be associated with tissue tropism and pathogenicity. C. psittaci harbors 21 Pmps. We hypothesize that the different infectious potential and host tropism of avian and mammalian C. psittaci strains is dependent on differences in their Pmp repertoires. In this study, we experimentally confirmed the different virulence of avian and mammalian strains, by testing the survival rate of infected embryonated eggs and chlamydiae dissemination in the embryos. Further, we investigated the possible involvement of Pmps in host tropism. Analysis of pmp sequences from 10 C. psittaci strains confirmed a high degree of variation, but no correlation with host tropism was identified. However, comparison of Pmp expression profiles from different strains showed that Pmps of the G group are the most variably expressed, also among avian and mammalian strains. To investigate their functions, selected Pmps were recombinantly produced from one avian and one mammalian representative strain and their adhesion abilities and relevance for the infection of C. psittaci strains in avian and mammalian cells were tested. For the first time, we identified Pmp22D, Pmp8G, and OmcB as relevant adhesins, essential during infection of C. psittaci strains in general. Moreover, we propose Pmp17G as a possible key player for host adaptation, as it could only bind to and influence the infection in avian cells, but it had no relevant impact towards infection in mammalian cells. These data support the hypothesis that distinct Pmp repertoires in combination with specific host factors may contribute to host tropism of C. psittaci strains.

Project description:The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions.IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

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Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

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