Type VI secretion system mutations reduced competitive fitness of classical Vibrio cholerae biotype.

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

IFN and cytokine responses in ducks to genetically similar H5N1 influenza A viruses of varying pathogenicity.

Ducks, the reservoir host, are generally permissive to influenza A virus infection without disease symptoms. This natural ecology was upset by the emergence of H5N1 strains, which can kill ducks. To better understand host-virus interactions in the reservoir host, and influenza strain-specific molecular contributions to virulence, we infected White Pekin ducks with three similar H5N1 viruses, with known differences in pathogenicity and replication rate. We quantified viral replication and innate immune gene activation by qPCR, in lung and spleen tissues, isolated on each of the first 3 days of infection. The three viruses replicated well, as measured by accumulation of matrix gene transcript, and viral load declined over time in the spleen. The ducks produced rapid, but temporally limited, IFN and cytokine responses, peaking on the first day post-infection. IFN and proinflammatory cytokine gene induction were greater in response to infection with the more lethal viruses, compared to an attenuated strain. We conclude that a well-regulated IFN response, with the ability to overcome early viral immune inhibition, without hyperinflammation, contributes to the ability of ducks to survive H5N1 influenza replication in their airways, and yet clear systemic infection and limit disease.

Extensive Allelic Diversity of MHC Class I in Wild Mallard Ducks.

MHC class I is critically involved in defense against viruses, and diversity from polygeny and polymorphism contributes to the breadth of the immune response and health of the population. In this article, we examine MHC class I diversity in wild mallard ducks, the natural host and reservoir of influenza A viruses. We previously showed domestic ducks predominantly use UAA, one of five MHC class I genes, but whether biased expression is also true for wild mallards is unknown. Using RT-PCR from blood, we examined expressed MHC class I alleles from 38 wild mallards (Anas platyrhynchos) and identified 61 unique alleles, typically 1 or 2 expressed alleles in each individual. To determine whether expressed alleles correspond to UAA adjacent to TAP2 as in domestic ducks, we cloned and sequenced genomic UAA-TAP2 fragments from all mallards, which matched transcripts recovered and allowed us to assign most alleles as UAA Allelic differences are primarily located in α1 and α2 domains in the residues known to interact with peptide in mammalian MHC class I, suggesting the diversity is functional. Most UAA alleles have unique residues in the cleft predicting distinct specificity; however, six alleles have an unusual conserved cleft with two cysteine residues. Residues that influence peptide-loading properties and tapasin involvement in chicken are fixed in duck alleles and suggest tapasin independence. Biased expression of one MHC class I gene may make viral escape within an individual easy, but high diversity in the population places continual pressure on the virus in the reservoir species.

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae.

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen's arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.

Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

Isolation and cytochemical characterization of melanomacrophages and melanomacrophage clusters from goldfish (Carassius auratus, L.).

Pigmented or "melano-" macrophages are prominent in lymphoid and non-lymphoid tissues of poikilotherms. Though they have been extensively studied in situ only recently has a means to isolate them from other cell types been established. We provide the first in vitro characterization of isolated melanomacrophage cytochemistry and survival in culture. Unlike non-pigmented tissue macrophages melanomacrophages do not adhere to polystyrene surfaces making them easy to separate from tissue macrophages. In vitro goldfish melanomacrophages are distinguishable from tissue macrophages and neutrophils by being Sudan Black B positive (unlike tissue macrophages) and non-specific esterase positive (unlike neutrophils). Like tissue macrophages they also express acid phosphatase and CSF-1R. As sorted cells melanomacrophages only survive a few days in culture. However in coarsely disaggregated spleen and kidney tissues melanomacrophages survive for at least 3 weeks. Furthermore after 5 days culture disaggregating tissue clumps revealed encapsulated melanomacrophage clusters that remained intact for at least another week. The encapsulated clusters were resilient enough to allow for their isolation for further imaging and isolation of RNA. In some cases the clusters had either melanomacrophages or non-fluorescent cells protruding and in the latter case these could initiate outgrowths onto the plates with subsequent collapse of the cluster. These approaches for the isolation of melanomacrophages and melanomacrophage clusters should allow further study into specific cell and cluster functions.