Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N‐terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical‐scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide‐conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti‐aggregation assays and size‐exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR‐conjugated AuNPs, which exhibit concentration‐dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp‐AuNPs in rescuing aberrant protein aggregation.
OBJECTIVE
To create antibiograms for commonly cultured organisms in a small animal tertiary care hospital following Clinical and Laboratory Standards Institute guidelines and to compare these local resistance patterns to published first-tier antimicrobial recommendations.
SAMPLE
Urine (n = 429), respiratory (41), and skin (75) isolates cultured from dogs between January 1, 2019, and December 31, 2020, at the Tufts University Foster Hospital for Small Animals.
PROCEDURES
MIC and susceptibility interpretations were recorded for multiple sites for 2 years. Sites with greater than 30 isolates for at least 1 organism were included. Urinary, respiratory, and skin antibiograms were created using Clinical and Laboratory Standards Institute breakpoints and guidelines.
RESULTS
Urinary Escherichia coli had a higher susceptibility percentage for amoxicillin–clavulanate (80% [221/275]) than amoxicillin alone (64% [175/275]). Respiratory E coli were greater than 80% susceptible to only 2 antimicrobials (imipenem, amikacin). Of skin Staphylococcus pseudintermedius isolates, 40% (30/75) were methicillin-resistant and frequently also displayed resistance to non-beta lactam antimicrobials. Susceptibility to recommended first-line antimicrobials varied and was greatest for gram-negative urinary isolates and lowest for methicillin-resistant S pseudintermedius skin isolates and respiratory E coli.
CLINICAL RELEVANCE
Local antibiogram creation identified frequent resistance that may preclude the use of guideline-recommended first-line therapy. High levels of resistance identified in methicillin-resistant S pseudintermedius isolates supports growing concern for methicillin-resistant staphylococci in veterinary patients. This project highlights the need for population-specific resistance profiles to be used in conjunction with national guidelines.
Bacteria use non‐coding small RNAs (sRNAs) to regulate expression of target mRNAs, often with the help of protein chaperones. About half of bacterial species possess a well‐characterized globally‐acting RNA‐binding protein, a hexameric protein called Hfq, which contributes to virulence in many pathogenic species. While much is known about the mechanism of RNA interactions by Hfq proteins in certain well‐studied model bacteria, there are significant gaps in our understanding of how conserved the RNA‐binding behavior of Hfq homologs from diverse bacterial organisms. Here, we use a recently developed bacterial three‐hybrid (B3H) assay to further define the interactions between Hfq and its RNA substrates. This assay connects the interaction between a DNA‐bound RNA and a RNA polymerase (RNAP)‐bound protein Hfq to the expression of a reporter gene, providing a genetic route to the analysis of RNA interactions from a heterologously expressed RNA‐binding protein. This provides a facile route to study the behavior of diverse RNA‐binding proteins in an innocuous laboratory strain of E. coli. Here we present data collected using the B3H assay to compare the RNA interactions of Hfq from several bacterial species, including Listeria monocytogenes, Caulobacter crescentus, and Escherichia coli. We have observed distinct RNA‐binding profiles between these Hfq proteins. Sequence alignments of these three proteins led to a hypothesis that charged residues along the rim surface of Hfq may underlie the differences in binding preferences for different sRNAs. Data will be presented that test this model using both chimeric and site‐directed mutants. Our long‐term goal is to gain a better understanding of what structural elements of an RNA‐binding protein such as Hfq determine its RNA binding and function in driving bacterial gene regulation.
Support or Funding Information
This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
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