Bovine Coronavirus Infects the Respiratory Tract of Cattle Challenged Intranasally.

Bovine Coronavirus (BCoV) is a member of a family of viruses associated with both enteric and respiratory diseases in a wide range of hosts. BCoV has been well-established as a causative agent of diarrhea in cattle, however, its role as a respiratory pathogen is controversial. In this study, fifteen calves were challenged intranasally with virulent BCoV in order to observe the clinical manifestation of the BCoV infection for up to 8 days after initial challenge, looking specifically for indication of symptoms, pathology, and presence of viral infection in the respiratory tract, as compared to six unchallenged control calves. Throughout the study, clinical signs of disease were recorded and nasal swabs were collected daily. Additionally, bronchoalveolar lavage (BAL) was performed at 4 days Post-challenge, and blood and tissue samples were collected from calves at 4, 6, or 8 days Post-challenge to be tested for the presence of BCoV and disease pathology. The data collected support that this BCoV challenge resulted in respiratory infections as evidenced by the isolation of BCoV in BAL fluids and positive qPCR, immunohistochemistry (IHC), and histopathologic lesions in the upper and lower respiratory tissues. This study can thus be added to a growing body of data supporting that BCoV is a respiratory pathogen and contributor to respiratory disease in cattle.

Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability.

Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis.

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis.

Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ54) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ54 transcriptional activation. CtcC activation of σ54 requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ54 transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ54 promoter elements. Reporter gene analyses using these putative σ54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ54 controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies.IMPORTANCE The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia, sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells.

Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis support a role in TCA cycle regulation.

Chlamydia trachomatis is an obligate intracellular bacteria that undergo dynamic morphologic and physiologic conversions upon gaining an access to a eukaryotic cell. These conversions likely require the detection of key environmental conditions and regulation of metabolic activity. Chlamydia encodes homologs to proteins in the Rsb phosphoregulatory partner-switching pathway, best described in Bacillus subtilis. ORF CT588 has a strong sequence similarity to RsbU cytoplasmic phosphatase domain but also contains a unique periplasmic sensor domain that is expected to control the phosphatase activity. A 1.7 Å crystal structure of the periplasmic domain of the RsbU protein from C. trachomatis (PDB 6MAB) displays close structural similarity to DctB from Vibrio and Sinorhizobium. DctB has been shown, both structurally and functionally, to specifically bind to the tricarboxylic acid (TCA) cycle intermediate succinate. Surface plasmon resonance and differential scanning fluorimetry of TCA intermediates and potential metabolites from a virtual screen of RsbU revealed that alpha-ketoglutarate, malate and oxaloacetate bound to the RsbU periplasmic domain. Substitutions in the putative binding site resulted in reduced binding capabilities. An RsbU null mutant showed severe growth defects which could be restored through genetic complementation. Chemical inhibition of ATP synthesis by oxidative phosphorylation phenocopied the growth defect observed in the RsbU null strain. Altogether, these data support a model with the Rsb system responding differentially to TCA cycle intermediates to regulate metabolism and key differentiation processes.