The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Comprehensive Assessment of Subtyping Methods for Improved Surveillance of Foodborne Salmonella.

High-resolution and efficient typing for the bacterial pathogen is essential for tracking the sources, detecting or diagnosing variants, and conducting a risk assessment. However, a systematic in-field investigation of Salmonella along the food chain has not been documented. This study assessed 12 typing methods, such as antimicrobial-resistance (AMR) gene profile typing, Core Genome Multilocus Sequence Typing (cgMLST), and CRISPR multi-virulence locus sequence typing (CRISPR-MVLST), to evaluate their effectiveness for use in routine monitoring of foodborne Salmonella transmission along the poultry production chain. During 2015-16, a total of 1,064 samples were collected from poultry production chain, starting from breeding farms and slaughterhouses to the markets of Zhejiang province in China. A total of 61 consecutive unique Salmonella isolates recovered from these samples were selected for genome sequencing and further comparative typing analysis. Traditional typing methods, including serotyping, AMR phenotype-based typing, as well as modern genotyping approaches, were evaluated and compared by their discrimination index (DI). The results showed that the serotyping method identified nine serovars. The gold standard cgMLST method indicated only 18 different types (DI = 0.8541), while the CRISPR-MVLST method detected 30 types (DI = 0.9628), with a higher DI than all examined medium-resolution WGS-based genotyping methods. We demonstrate that the CRISPR-MVLST might be used as a tool with high discriminatory power, comparable ease of use, ability of tracking the source of Salmonella strains along the food chain and indication of genetic features especially virulence genes. The available methods with different purposes and laboratory expertise were also illustrated to assist in rational implementation. IMPORTANCE In public health field, high-resolution and efficient typing of the bacterial pathogen is essential, considering source-tracking and risk assessment are fundamental issues. Currently, there are no recommendations for applying molecular characterization methods for Salmonella along the food chain, and a systematic in-field investigation comparing subtyping methods in the context of routine surveillance was partially addressed. Using 1,064 samples along a poultry production chain with a considerable level of Salmonella contamination, we collected representative isolates for genome sequencing and comparative analysis by using 12 typing techniques, particularly with whole-genome sequence (WGS) based methods and a recently invented CRISPR multi-virulence locus sequence typing (CRISPR-MVLST) method. CRISPR-MVLST is identified as a tool with higher discriminatory power compared with medium-resolution WGS-based typing methods, comparable ease of use and proven ability of tracking Salmonella isolates. Besides, we also offer recommendations for rational choice of subtyping methods to assist in better implementation schemes.

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

PrsA contributes to Streptococcus suis serotype 2 pathogenicity by modulating secretion of selected virulence factors.

Streptococcus suis serotype 2 (S. suis 2) is a major zoonotic pathogen. Parvulin-type peptidyl-prolyl isomerase (PrsA) in S. suis 2 is found surface-associated, pro-inflammatory and cytotoxic. To further explore the roles of PrsA in S. suis 2 infection, we constructed a prsA deletion mutant (ΔprsA) and a complemented strain (CΔprsA). The ΔprsA mutant showed increased length of bacterial chains and decreased growth. Deletion of prsA increased bacterial adhesion to host epithelial cells but with weakened invasion. The ΔprsA mutant had reduced survival in RAW264.7 macrophages and pig whole blood, and significantly attenuated in virulence to mice. All these phenotypes of the mutant could be reversed largely to the levels of its parental strain by gene complementation. Western blotting revealed that suilysin was markedly reduced both in surface-associated (SAP) and secreted fractions (SecP) of ΔprsA, which might be responsible for reduced hemolytic activity. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase were significantly increased in both SAP and SecP fractions as a result of prsA deletion. Increased adhesion of the ΔprsA mutant to bEND.3 cells was prevented using polyclonal antibodies against GAPDH and enolase. Overall, we propose that S. suis 2 deploys PrsA to control translocation of important virulence factors, thereby favoring its survival in the host with enhanced pathogenicity by compromising its interactions with the host cells. Further investigation is required to find out how PrsA modulates protein translocation to benefit S. suis infection and if there are other S. suis 2 substrates of potential virulence regulated by PrsA.

Peptidyl isomerase PrsA is surface-associated on Streptococcus suis and offers cross-protection against serotype 9 strain.

PrsA, a peptidyl isomerase encoded by the prsA gene, plays pleiotropic roles in bacterial physiology and pathogenicity. This study was attempted to characterize the distribution of prsA in different serotypes of Streptococcus suis isolates and on the bacterial cells and to evaluate its immunogenicity in a murine model. PrsA is present in different S. suis types and surface-associated as tested in a S. suis type 2 strain. The prsA gene from the serotype 2 strain was cloned for its expression in Escherichia coli. Recombinant PrsA (SsPrsA) showed good reactivity with anti-sera to S. suis serotype 2 and 9 strains. Immunization of mice with SsPrsA elicited a significant antibody response and conferred partial protection against lethal challenge with a S. suis serotype 2 strain (50% protection) or a serotype 9 strain (66% protection). The anti-SsPrsA sera showed good reactivity to the surface-associated proteins of both serotype 2 and serotype 9 strains. Higher abundance of surface-associated PrsA in the serotype 9 strain (than the serotype 2 strain) might account, in part, for higher protection against its challenge infection. These results suggest that SsPrsA may serve as a novel subunit vaccine candidate with cross-protective potential.

A Meta-Analysis of Major Foodborne Pathogens in Chinese Food Commodities Between 2006 and 2016.

Prevalence of pathogenic bacteria in food commodities in China have been reported in numerous publications over time. However, the results are scattered and varied. To calculate a robust point estimate with a higher statistical power, we applied meta-analytic approach for investigating the prevalence of common foodborne pathogens in major food items in China. Data, on prevalence of bacteria in various food commodities were extracted and analyzed from 361 (132 English and 229 Chinese) publications. Prevalence of eight most frequently reported pathogens on six broad food categories was used for pooled and subgroup meta-analysis by DerSimonian-Laird method in random-effects model. The estimated overall prevalence of pathogens in the foods was 8.5% (95% CI 8.2-8.7). The highest prevalence, irrespective of the pathogen type, was in the aquatic produce at 12.8% (12.0-13.5), while the least was in the vegetables at 3.0% (2.6-3.4). Among the pathogens, the most prevalent was Vibrio at 21.3% (19.6-23.1), whereas the least was pathogenic Escherichia coli at 4.3% (3.3-5.2). The major food pathogens in Chinese foods in decreasing order of prevalence were Vibrio parahaemolyticus, Campylobacter, Bacillus cereus, Staphylococcus aureus, Salmonella, Enterobacter, Listeria monocytogenes, and pathogenic E. coli. Presence of these organisms in foods equates the risk of microbiological food safety in China with other developed countries rather than the developing countries. This justifies the need of novel perspectives for formulating policies on microbiological food safety and risk mitigation.