Biology subjects

Haag, A.

Publications and source records attributed to Haag, A..

3 recordsLinked to original sources

bioRxiv2024

Clostridioides difficile colonization is not mediated by bile salts and requires Stickland fermentation of proline in an in vitro model of infection

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic- treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (prdB-) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. Inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile. IMPORTANCEC. difficile is one of the leading causes of hospital-acquired infections and antibiotic-associated diarrhea. Several potential mechanisms through which the microbiota can limit C. difficile infection have been identified and are potential targets for new therapeutics. However, it is unclear which mechanisms of C. difficile inhibition represent the best targets for development of new therapeutics. These studies demonstrate that in a complex in vitro model of C. difficile infection, colonization resistance is independent of microbial bile salt metabolism. Instead, the ability of C. difficile to colonize is dependent upon its ability to metabolize proline, although proline-dependent colonization is context-dependent and is not observed in all disrupted communities. Altogether, these studies support the need for further work to understand how bile- independent mechanisms regulate C. difficile colonization.

microbiology↗

bioRxiv2020

A Glu-Glu-Tyr sequence in the cytoplasmic tail of the M2 protein renders IAV susceptible to restriction of HA-M2 association in primary human macrophages

Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes although particle formation itself requires only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDM) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. The defects in HA-M2 association and particle assembly are specific to MDM and not observed in a monocytic cell line differentiated into macrophage-like cells. Notably, both these defects can be reversed upon disruption of the actin cytoskeleton. In the current study, we sought to examine whether M2 contributes to particle assembly in MDM and to identify a viral determinant involved in the MDM-specific and actin-dependent suppression of IAV assembly. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus fails to form budding structures at the cell surface even after F-actin is disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74-76) in the M2 cytoplasmic tail allows HA-M2 association to occur efficiently even in MDM with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identifies a viral determinant for susceptibility to cytoskeleton-dependent regulation in MDM and hence, sheds light on the molecular mechanism behind the MDM-specific restriction of IAV assembly. ImportanceNon-permissive cell types that are unable to support viral replication serve as important tools for identification of host factors that either block viral replication (restriction factors) or support viral replication in permissive cell types (co-factors). We previously identified the MDM as a cell type that is non-permissive to IAV assembly, likely due to a block in HA-M2 association. In the current study, we determined that the IAV M2 protein is necessary for virus particle formation in MDM but also renders the virus susceptible to the MDM-specific suppression of virus assembly. We identified a specific amino acid motif in the M2 cytoplasmic tail, disruption of which allows M2 to associate with HA even in MDM. Our findings strongly support the possibility that the MDM-specific defect in HA-M2 association is due to the presence of a restriction factor(s) in MDM, which likely interacts directly with the M2 cytoplasmic domain, rather than indirectly through other internal viral components, and thereby prevents M2 from associating with HA.

microbiology↗

bioRxiv2019

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and human cells

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. The putative co-chaperone CHP-1 acts as a regulator of EGFR trafficking during C.elegans vulval development. Loss of chp-1 causes the retention of the EGFR in the ER and decreased MAPK signaling. CHP-1 functions specifically, as the localization of other receptors is unaltered in chp-1(lf) mutants, and inhibiting other co-chaperones does not affect EGFR localization. The role of CHP-1 during EGFR trafficking is conserved in humans. Analogous to C.elegans, the response of CHP-1-deficient human cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity is decreased. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate an HSP90-independent function of CHP-1. The identification of CHP-1 as a regulator of EGFR trafficking opens the possibility to identify small molecule chaperone inhibitors targeting the EGFR pathway with increased selectivity.

cell biology↗