Yersinia spp. vary significantly in their ability to cause diseases that threaten public health. Their pathogenicity is frequently associated with increasing antimicrobial resistance (AMR) and various virulence factors. The aim of the study was to investigate the AMR genes, virulence factors, and genetic diversity of Yersinia strains isolated from meats and fish in Wenzhou in 2020 by using whole-genome sequencing (WGS). A total of 50 isolates were collected. The phylogenetic relationships among the Yersinia species were also analyzed using multilocus sequence typing (MLST), core genome multi-locus sequence typing (cgMLST), and single nucleotide polymorphism (SNP) analysis. According to the results, all the strains could be classified into five species, with most isolated from beef, followed by poultry, pork, and fish. AMR genes were identified in 23 strains. And the qnrD1 genes were all located in the Col3M plasmid. Virulence genes, such as yaxA, ystB, pla, and yplA, were also found in the 15 Y. enterocolitica strains. And this study also found the presence of icm/dot type IVB-related genes in one Yersinia massiliensis isolate. MLST analysis identified 43 sequence types (STs), 19 of which were newly detected in Yersinia. Moreover, cgMLST analysis revealed that no dense genotype clusters were formed (cgMLST 5341, 5344, 5346-5350, 5353-5390). Instead, the strains appeared to be dispersed over large distances, except when multiple isolates shared the same ST. Isolates Y4 and Y26 were closely related to strains originating from South Korea and Denmark. This study showed considerable diversity in Yersinia spp. isolated from local areas (Wenzhou City). The data generated in our study may enrich the molecular traceability database of Yersinia and provide a basis for the development of more effective antipathogen control strategies.

Yersinia pestis is the etiological agent of plague. Marmota himalayana of the Qinghai-Tibetan plateau is the primary host of flea-borne Y. pestis. This study is the report of isolation of Mu-like bacteriophages of Y. pestis from M. himalayana. The isolation and characterization of four Mu-like phages of Y. pestis were reported, which were named as vB_YpM_3, vB_YpM_5, vB_YpM_6, and vB_YpM_23 according to their morphology. Comparative genome analysis revealed that vB_YpM_3, vB_YpM_5, vB_YpM_6, and vB_YpM_23 are phylogenetically closest to Escherichia coli phages Mu, D108 and Shigella flexneri phage SfMu. The role of LPS core structure of Y. pestis in the phages\' receptor was pinpointed. All the phages exhibit \"temperature dependent infection,\" which is independent of the growth temperature of the host bacteria and dependent of the temperature of phage infection. The phages lyse the host bacteria at 37°C, but enter the lysogenic cycle and become prophages in the chromosome of the host bacteria at 26°C. IMPORTANCE Mu-like bacteriophages of Y. pestis were isolated from M. himalayana of the Qinghai-Tibetan plateau in China. These bacteriophages have a unique temperature dependent life cycle, follow a lytic cycle at the temperature of warm-blooded mammals (37°С), and enter the lysogenic cycle at the temperature of its flea-vector (26°С). A switch from the lysogenic to the lytic cycle occurred when lysogenic bacteria were incubated from lower temperature to higher temperature (initially incubating at 26°C and shifting to 37°C). It is speculated that the temperature dependent lifestyle of bacteriophages may affect the population dynamics and pathogenicity of Y. pestis.

Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn2+ acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn2+ into the periplasm by the YezP-Zn2+ complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn2+ from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. IMPORTANCE Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn2+ acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn2+ into the cytoplasm via the YezP-Zn2+ complex, and elucidation of the complete Zn2+ acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0-11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases.

To colonize in the digestive tract of animals and humans, Yersinia pseudotuberculosis has to deal with reactive oxygen species (ROS) produced by host cells and microbiota. However, an understanding of the ROS-scavenging systems and their regulation in this bacterium remains largely elusive. In this study, we identified OxyR as the master transcriptional regulator mediating cellular responses to hydrogen peroxide (H2O2) in Y. pseudotuberculosis through genomics and transcriptomics analyses. OxyR activates transcription of diverse genes, especially the core members of its regulon, including those encoding catalases, peroxidases, and thiol reductases. The data also suggest that sulfur species and manganese may play a particular role in the oxidative stress response of Y. pseudotuberculosis. Among the three H2O2-scavenging systems in Y. pseudotuberculosis, catalase/peroxidase KatE functions as the primary scavenger for high levels of H2O2; NADH peroxidase alkyl hydroperoxide reductase (AhpR) and catalase KatG together are responsible for removing low levels of H2O2. The simultaneous loss of both AhpC (the peroxidatic component of AhpR) and KatG results in activation of OxyR. Moreover, we found that AhpC, unlike its well-characterized Escherichia coli counterpart, has little effect on protecting cells against toxicity of organic peroxides. These findings provide not only novel insights into the structural and functional diversity of bacterial H2O2-scavenging systems but also a basic understanding of how Y. pseudotuberculosis copes with oxidative stress.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a \"two-level\" evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

Apical sodium-dependent bile acid transporter (ASBT) retrieves bile acids from the small intestine and plays a pivotal role in enterohepatic circulation. Currently, high-resolution structures are available for two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii), from which an elevator-style alternating-access mechanism has been proposed for substrate transport. A key concept in this model is that the substrate binds to the central cavity of the transporter so that the elevator-like motion can expose the bound substrate alternatingly to either side of the membrane during a transport cycle. However, no structure of an ASBT has been solved with a substrate bound in its central cavity, so how a substrate binds to ASBT remains to be defined. In this study, molecular docking, structure determination and functional analysis were combined to define and validate the details of substrate binding in ASBTYf. The findings provide coherent evidence to provide a clearer picture of how the substrate binds in the central cavity of ASBTYf that fits the alternating-access model.

Apical sodium-dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator-style alternating-access model, which was deduced mainly by comparing high-resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward-facing structure (PDB entry 4n7x) was obtained with an Na+-binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+- or substrate-binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild-type ASBTYf. These data support the hypothesis that the PDB entry 4n7x-like structure represents a functional outward-facing conformation of ASBTYf in its transport cycle.

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to block the host innate immune response. One of the effectors, YopT, is a potent cysteine protease that causes the disruption of the actin cytoskeleton to inhibit phagocytosis of the pathogen; however, its molecular mechanism and relevance to pathogenesis need further investigation. In this report, we show that RIG-I is a novel target of the YopT protein. Remarkably, YopT interacts with RIG-I and inhibits rat liver homogenate-mediated nuclear factor-κB and interferon regulatory factor-3 activation. Further studies revealed a YopT-dependent increase in the K48-polymerized ubiquitination of RIG-I. These findings suggest that YopT negatively regulates RIG-I-mediated cellular antibacterial response by targeting RIG-I.

Microbial pathogens have evolved complex mechanisms to interface with host cells in order to evade host defenses and replicate. However, mammalian innate immune receptors detect the presence of molecules unique to the microbial world or sense the activity of virulence factors, activating antimicrobial and inflammatory pathways. We focus on how studies of the major virulence factor of one group of microbial pathogens, the type III secretion system (T3SS) of human pathogenic Yersinia, have shed light on these important innate immune responses. Yersinia are largely extracellular pathogens, yet they insert T3SS cargo into target host cells that modulate the activity of cytosolic innate immune receptors. This review covers both the host pathways that detect the Yersinia T3SS and the effector proteins used by Yersinia to manipulate innate immune signaling.