A Yeast Two-Hybrid Screen for SYP-3 Interactors Identifies SYP-4, a Component Required for Synaptonemal Complex Assembly and Chiasma Formation in Caenorhabditis elegans Meiosis

The proper assembly of the synaptonemal complex (SC) between homologs is critical to ensure accurate meiotic chromosome segregation. The SC is a meiotic tripartite structure present from yeast to humans, comprised of proteins assembled along the axes of the chromosomes and central region (CR) proteins that bridge the two chromosome axes. Here we identify SYP-4 as a novel structural component of the SC in Caenorhabditis elegans. SYP-4 interacts in a yeast two-hybrid assay with SYP-3, one of components of the CR of the SC, and is localized at the interface between homologs during meiosis. SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes. In the absence of SYP-4, the levels of recombination intermediates, as indicated by RAD-51 foci, are elevated in mid-prophase nuclei, and crossover recombination events are significantly reduced. The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality. Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly

CRA-1 Uncovers a Double-Strand Break-Dependent Pathway Promoting the Assembly of Central Region Proteins on Chromosome Axes During C. elegans Meiosis

The synaptonemal complex (SC), a tripartite proteinaceous structure that forms between homologous chromosomes during meiosis, is crucial for faithful chromosome segregation. Here we identify CRA-1, a novel and conserved protein that is required for the assembly of the central region of the SC during C. elegans meiosis. In the absence of CRA-1, central region components fail to extensively localize onto chromosomes at early prophase and instead mostly surround the chromatin at this stage. Later in prophase, central region proteins polymerize along chromosome axes, but for the most part fail to connect the axes of paired homologous chromosomes. This defect results in an inability to stabilize homologous pairing interactions, altered double-strand break (DSB) repair progression, and a lack of chiasmata. Surprisingly, DSB formation and repair are required to promote the polymerization of the central region components along meiotic chromosome axes in cra-1 mutants. In the absence of both CRA-1 and any one of the C. elegans homologs of SPO11, MRE11, RAD51, or MSH5, the polymerization observed along chromosome axes is perturbed, resulting in the formation of aggregates of the SC central region proteins. While radiation-induced DSBs rescue this polymerization in cra-1; spo-11 mutants, they fail to do so in cra-1; mre-11, cra-1; rad-51, and cra-1; msh-5 mutants. Taken together, our studies place CRA-1 as a key component in promoting the assembly of a tripartite SC structure. Moreover, they reveal a scenario in which DSB formation and repair can drive the polymerization of SC components along chromosome axes in C. elegans

Genes Activated by Vibrio cholerae upon Exposure to Caenorhabditis elegans Reveal the Mannose-Sensitive Hemagglutinin To Be Essential for Colonization

The waterborne diarrheal disease cholera is caused by the bacterium Vibrio cholerae. The facultative human pathogen persists as a natural inhabitant in the aquatic ecosystem between outbreaks. In contrast to the human host, V. cholerae requires a different set of genes to survive in this hostile environment. For example, predatory micrograzers are commonly found in the aquatic environment and use bacteria as a nutrient source, but knowledge of the interaction between bacterivorous grazers and V. cholerae is limited. In this study, we successfully adapted a genetic reporter technology and identified more than 100 genes activated by V. cholerae upon exposure to the bacterium-grazing nematode Caenorhabditis elegans. This screen provides a first glimpse into responses and adaptational strategies of the bacterial pathogen against such natural predators. Subsequent phenotypic characterization revealed the mannose-sensitive hemagglutinin to be crucial for colonization of the worm, which causes developmental delay and growth retardation.During its life cycle, the facultative human pathogen Vibrio cholerae, which is the causative agent of the diarrheal disease cholera, needs to adapt to a variety of different conditions, such as the human host or the aquatic environment. Importantly, cholera infections originate from the aquatic reservoir where V. cholerae persists between the outbreaks. In the aquatic environment, bacteria are constantly threatened by predatory protozoa and nematodes, but our knowledge of the response pathways and adaptation strategies of V. cholerae to such stressors is limited. Using a temporally controlled reporter system of transcription, we identified more than 100 genes of V. cholerae induced upon exposure to the nematode Caenorhabditis elegans, which emerged recently as a valuable model for environmental predation during the aquatic lifestyle of V. cholerae. Besides others, we identified and validated the genes encoding the mannose-sensitive hemagglutinin (MSHA) type IV pilus to be significantly induced upon exposure to the nematode. Subsequent analyses demonstrated that the mannose-sensitive hemagglutinin is crucial for attachment of V. cholerae in the pharynx of the worm and initiation of colonization, which results in growth retardation and developmental delay of C. elegans. Thus, the surface adhesion factor MSHA could be linked to a fitness advantage of V. cholerae upon contact with bacterium-grazing nematodes

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections

Characterization of the immune response from mice intraperitoneally immunized with NTHi OMVs.

<p>(A) Shown are the median IgG1and IgA titers to OMVs derived from NTHi strain 2019-R in sera from mice intraperitoneally immunized with IM-1 collected at day 0 and 39 (n = 7). (B) Shown are the median half-maximum total Ig titers to OMVs derived from NTHi strains 2019-R in sera collected at day 39 from mice intraperitoneally immunized with IM-1 (i.p. IM-1) as well as from nonvaccinated control mice (co) (n = 6 for the co group and n = 7 for the i.p. IM-1 group). The error bars indicate the interquartile range of each data set for each time point. Significant differences between the data sets are marked by asterisks (<i>P</i><0.05; Mann-Whitney U test).</p

Induced immune responses in mice intranasally immunized with NTHi OMVs are protective against nasopharyngeal challenge.

<p>Shown are the nasopharyngeal colonization rates in recovered CFU per nasopharynx for mice intranasally immunized with either IM-1 or IM-2 as well as for nonvaccinated control mice (co). Mice were intranasally challenged with either NTHi strain 2019-R (A) or 3198-R (B) for 24 h. Each circle represents the recovered CFU per nasopharynx from one mouse. The horizontal bars indicate the median of each data set. If no bacteria could be recovered, then the values were set to the limit of detection of 10 CFU/nasopharynx (indicated by the dotted line). Since independent immunization rounds were performed, the exact infection doses ranged from 3.3×10⁵ to 6.0×10⁵ CFU/mouse for NTHi strain 2019-R and from 4.1×10⁵ to 4.3×10⁵ CFU/mouse for NTHi strain 3198-R. Significant differences between the data sets are marked by asterisks (<i>P</i><0.05; Kruskal-Wallis test and <i>post hoc</i> Dunn's multiple comparisons).</p

Temporal immune responses to OMVs derived from NTHi strain 2019-R.

<p>Shown are the median titers over time of IgM (A), IgA (B), and IgG1 (C) antibodies to 2019-R OMVs in sera from mice intranasally immunized with either IM-1 (solid line) or IM-2 (dashed line) as well as in sera from nonvaccinated control mice (dotted line) (n = 20 for each group). The error bars indicate the interquartile range of each data set for each time point.</p