Loss to gain: pseudogenes in microorganisms, focusing on eubacteria, and their biological significance.

Pseudogenes are defined as "non-functional" copies of corresponding parent genes. The cognition of pseudogenes continues to be refreshed through accumulating and updating research findings. Previous studies have predominantly focused on mammals, but pseudogenes have received relatively less attention in the field of microbiology. Given the increasing recognition on the importance of pseudogenes, in this review, we focus on several aspects of microorganism pseudogenes, including their classification and characteristics, their generation and fate, their identification, their abundance and distribution, their impact on virulence, their ability to recombine with functional genes, the extent to which some pseudogenes are transcribed and translated, and the relationship between pseudogenes and viruses. By summarizing and organizing the latest research progress, this review will provide a comprehensive perspective and improved understanding on pseudogenes in microorganisms. KEY POINTS: • Concept, classification and characteristics, identification and databases, content, and distribution of microbial pseudogenes are presented. • How pseudogenization contribute to pathogen virulence is highlighted. • Pseudogenes with potential functions in microorganisms are discussed.

FASN Inhibition Decreases MHC-I Degradation and Synergizes with PD-L1 Checkpoint Blockade in Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICI) transformed the treatment landscape of hepatocellular carcinoma (HCC). Unfortunately, patients with attenuated MHC-I expression remain refractory to ICIs, and druggable targets for upregulating MHC-I are limited. Here, we found that genetic or pharmacologic inhibition of fatty acid synthase (FASN) increased MHC-I levels in HCC cells, promoting antigen presentation and stimulating antigen-specific CD8+ T-cell cytotoxicity. Mechanistically, FASN inhibition reduced palmitoylation of MHC-I that led to its lysosomal degradation. The palmitoyltransferase DHHC3 directly bound MHC-I and negatively regulated MHC-I protein levels. In an orthotopic HCC mouse model, Fasn deficiency enhanced MHC-I levels and promoted cancer cell killing by tumor-infiltrating CD8+ T cells. Moreover, the combination of two different FASN inhibitors, orlistat and TVB-2640, with anti-PD-L1 antibody robustly suppressed tumor growth in vivo. Multiplex IHC of human HCC samples and bioinformatic analysis of The Cancer Genome Atlas data further illustrated that lower expression of FASN was correlated with a higher percentage of cytotoxic CD8+ T cells. The identification of FASN as a negative regulator of MHC-I provides the rationale for combining FASN inhibitors and immunotherapy for treating HCC. SIGNIFICANCE: Inhibition of FASN increases MHC-I protein levels by suppressing its palmitoylation and lysosomal degradation, which stimulates immune activity against hepatocellular carcinoma and enhances the efficacy of immune checkpoint inhibition.

The fexA gene in Campylobacter: whether the spread has occurred among various hosts in eastern China.

OBJECTIVES: The emergence of the florfenicol resistance gene fexA in Campylobacter poses a serious threat to public health, but the extent of the spread of fexA in Campylobacter from various hosts has not been well understood. This study aimed to investigate the fexA in Campylobacter isolates from different hosts. METHODS: PCR was used to identify fexA-positive Campylobacter from different hosts during 2008-2019 in China, and the fexA-positive isolates were characterized by susceptibility tests, whole-genome sequencing, and natural transformation. RESULTS: A total of 69 (2.54%, 69/2721) fexA-positive Campylobacter were identified, and the fexA-positive isolates increased remarkably (0.42%-16.90%) since it was first detected in 2010. By source, the 69 isolates were obtained from chickens (3.57%, 57/1595), geese (3.43%, 7/204), ducks (1.02%, 2/197), and environments (2.86%, 3/105); the fexA-positive isolates were not isolated in humans and pigs. In addition to fexA, these isolates also carried other antimicrobial resistance genes and exhibited multidrug resistance. Whole-genome sequencing analysis showed the fexA gene can disseminate clonally or horizontally via either multidrug resistance genomic islands or insertion sequences among the Campylobacter. The genetic structure IS1216-∆ISEfa11-hp-fexA-NAD(P)H-∆ISEfa11-IS1216 was conserved and widespread in the Campylobacter of various origins, and the IS1216 can form fexA-carrying circular intermediates, emphasizing that IS1216 plays an important role in the spread of fexA in Campylobacter. CONCLUSIONS: This study indicates the wide spread of fexA-positive Campylobacter in poultry and environments. Because multidrug resistance genomic islands and IS1216 can facilitate the transmission of fexA, systematic surveillance should be implemented to prevent the spread of fexA to humans.

Enhanced mitophagy driven by ADAR1-GLI1 editing supports the self-renewal of cancer stem cells in HCC.

BACKGROUND AND AIMS: Deregulation of adenosine-to-inosine editing by adenosine deaminase acting on RNA 1 (ADAR1) leads to tumor-specific transcriptome diversity with prognostic values for HCC. However, ADAR1 editase-dependent mechanisms governing liver cancer stem cell (LCSC) generation and maintenance have remained elusive. APPROACH AND RESULTS: RNA-seq profiling identified ADAR1-responsive recoding editing events in HCC and showed editing frequency of GLI1 , rather than transcript abundance was clinically relevant. Functional differences in LCSC self-renewal and tumor aggressiveness between wild-type (GLI1 wt ) and edited GLI1 (GLI1 edit ) were elucidated. We showed that overediting of GLI1 induced an arginine-to-glycine (R701G) substitution, augmenting tumor-initiating potential and exhibiting a more aggressive phenotype. GLI1 R701G harbored weak affinity to SUFU, which in turn, promoted its cytoplasmic-to-nuclear translocation to support LCSC self-renewal by increased pluripotency gene expression. Moreover, editing predisposed to stabilize GLI1 by abrogating ß-TrCP-GLI1 interaction. Integrative analysis of single-cell transcriptome further revealed hyperactivated mitophagy in ADAR1-enriched LCSCs. GLI1 editing promoted a metabolic switch to oxidative phosphorylation to control stress and stem-like state through PINK1-Parkin-mediated mitophagy in HCC, thereby conferring exclusive metastatic and sorafenib-resistant capacities. CONCLUSIONS: Our findings demonstrate a novel role of ADAR1 as an active regulator for LCSCs properties through editing GLI1 in the highly heterogeneous HCC.

Prevalence and genetic characterization of Campylobacter from clinical poultry cases in China.

IMPORTANCE: Campylobacter is a major cause of campylobacteriosis worldwide, and poultry is the main reservoir for its transmission. Campylobacter was generally considered to be a harmless commensal organism in poultry without pathogenic properties. However, it was proposed that a Campylobacter-like organism may be the cause of vibrionic hepatitis, which poses a significant public health risk. The occurrence and epidemiology of Campylobacter in healthy poultry have been studied systematically, but little is known about the epidemiology of Campylobacter isolates from diseased poultry in China. Therefore, this study determined the prevalence and molecular characterization of Campylobacter from diseased chickens, ducks, and geese in Yangzhou Veterinary Hospital between December 2016 and September 2017, which was critical for improving the diagnosis and prevention of Campylobacter infections.

Targeting MFGE8 secreted by cancer-associated fibroblasts blocks angiogenesis and metastasis in esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) play vital roles in establishing a suitable tumor microenvironment. In this study, RNA sequencing data revealed that CAFs could promote cell proliferation, angiogenesis, and ECM reconstitution by binding to integrin families and activating PI3K/AKT pathways in esophageal squamous cell carcinoma (ESCC). The secretions of CAFs play an important role in regulating these biological activities. Among these secretions, we found that MFGE8 is specifically secreted by CAFs in ESCC. Additionally, the secreted MFGE8 protein is essential in CAF-regulated vascularization, tumor proliferation, drug resistance, and metastasis. By binding to Integrin αVß3/αVß5 receptors, MFGE8 promotes tumor progression by activating both the PI3K/AKT and ERK/AKT pathways. Interestingly, the biological function of MFGE8 secreted by CAFs fully demonstrated the major role of CAFs in ESCC and its mode of mechanism, showing that MFGE8 could be a driver factor of CAFs in remodeling the tumor environment. In vivo treatment targeting CAFs-secreting MFGE8 or its receptor produced significant inhibitory effects on ESCC growth and metastasis, which provides an approach for the treatment of ESCC.

Evidence of potential Campylobacter jejuni zooanthroponosis in captive macaque populations.

Non-human primates share recent common ancestry with humans and exhibit comparable disease symptoms. Here, we explored the transmission potential of enteric bacterial pathogens in monkeys exhibiting symptoms of recurrent diarrhoea in a biomedical research facility in China. The common zoonotic bacterium Campylobacter jejuni was isolated from macaques (Macaca mulatta and Macaca fascicularis) and compared to isolates from humans and agricultural animals in Asia. Among the monkeys sampled, 5 % (44/973) tested positive for C. jejuni, 11 % (5/44) of which displayed diarrhoeal symptoms. Genomic analysis of monkey isolates, and 1254 genomes from various sources in Asia, were used to identify the most likely source of human infection. Monkey and human isolates shared high average nucleotide identity, common MLST clonal complexes and clustered together on a phylogeny. Furthermore, the profiles of putative antimicrobial resistance genes were similar between monkeys and humans. Taken together these findings suggest that housed macaques became infected with C. jejuni either directly from humans or via a common contamination source.

Evaluation of Hygiene Practice for Reducing Campylobacter Contamination on Cutting Boards and Risks Associated with Chicken Handling in Kitchen Environment.

Cutting boards can serve as potential carriers for the cross-contamination of pathogens from chicken to other surfaces. This study aimed to assess chefs' handling practices of cutting boards across five provinces in China and identify the key factors contributing to unsafe cutting board usage, including cleaning methods and handling practices. Handling practices associated with cutting boards were examined through a web-based survey (N = 154), while kitchen environment tests were conducted to investigate the splashing or survival of Campylobacter, inoculated in chicken or on cutting boards, to mimic the practices of chefs. Among chefs in the five provinces of China, wood and plastic cutting boards were the most commonly used for preparing chicken meat. Approximately 33.7% of chefs washed boards with running tap water, 31.17% of chefs washed boards with detergent, and 24.03% of chefs cleaned boards by scraping them with a knife after preparing other meats or chicken. The study tested 23 cutting boards from commercial kitchens for Campylobacter presence before and after chicken preparation and cleaning. Among these, 17 were cleaned with a knife, 5 with running tap water, and only 1 with disinfectant. Results showed that cleaning with a knife significantly reduced Campylobacter presence on cutting boards (p < 0.05), while the three main cleaning methods were inadequate in eliminating contamination to a safe level. In kitchen environment tests, contaminated chicken was chopped on cutting boards, with a maximum distance of 60 cm for low contamination, and 120 cm for medium and high contamination levels. This suggested a contamination risk exposure area ranging from 60 cm to 120 cm. Campylobacter survival on surfaces of wood, plastic, and stainless steel was also tested, with plastic surfaces showing the longest survival time (4.5 h at 15 °C and 3.5 h at 25 °C) In comparison, survival time on stainless steel or wood surfaces was only 3 h, implying a cross-contamination risk exposure period of 3 to 4.5 h after chicken preparation. In conclusion, based on the current study data, the practices employed by chefs play an important role in Campylobacter transfer in the kitchen environment. The presence of Campylobacter on cutting boards even after wiping or droplet splashing highlights its potential as a source of cross-contamination in the kitchen environment. So, chefs in China should reinforce their hygiene culture and adopt effective cutting board cleaning practices to prevent pathogen contamination.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

Genomic Insights into Pangenome and Antimicrobial Resistance in Campylobacter spp. Isolated from Chickens at Specific Growth Stages.

Improved understanding of the genetic basis of Campylobacter spp. colonization of poultry at specific growth stage is the key to developing a farm-based strategy to prevent flock colonization. In this study, 39 Campylobacter spp. strains (chicken isolates, n = 29; environmental isolates, n = 10) were collected from six marked chickens at the growth stage from week 7 to week 13. Then, we use comparative genomics techniques to analyze the temporal genomic characteristics of Campylobacter spp. in individual chickens across a production cycle. Genotype, average nucleotide identity (ANI), and phylogenetic trees all indicated the evolutionary relationships between the strains from different sampling weeks. The clustering of isolates was not dependent on sampling time and sample source, indicating that strains could persist over several weeks in a flock. Notably, 10 antimicrobial resistance (AMR) genes were identified in the genome of Campylobacter coli isolates, and the genomes of isolates sampled at week 11 harbored fewer AMR genes and insertion sequences (IS) than the isolates from other weeks. Consistent with this, pangenome-wide association analysis demonstrated that gene acquisition and loss could happen at week 11 and week 13. These genes were mainly associated with cell membrane biogenesis, ion metabolism, and DNA replication, suggesting that genomic change may be related to Campylobacter adaptive response. This is a novel study focused on the genetic changes occurring in Campylobacter spp. isolates in a particular space and time; it highlights that accessory genes and AMR genes were overall stable at chicken farm, which will help us understand the survival and the transmission route of Campylobacter spp. better, and have the potential to inform the strategy on the safety control of market-ready chickens.

Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction.

Despite the intense CD8+ T-cell infiltration in the tumor microenvironment of nasopharyngeal carcinoma, anti-PD-1 immunotherapy shows an unsatisfactory response rate in clinical trials, hindered by immunosuppressive signals. To understand how microenvironmental characteristics alter immune homeostasis and limit immunotherapy efficacy in nasopharyngeal carcinoma, here we establish a multi-center single-cell cohort based on public data, containing 357,206 cells from 50 patient samples. We reveal that nasopharyngeal carcinoma cells enhance development and suppressive activity of regulatory T cells via CD70-CD27 interaction. CD70 blocking reverts Treg-mediated suppression and thus reinvigorate CD8+ T-cell immunity. Anti-CD70+ anti-PD-1 therapy is evaluated in xenograft-derived organoids and humanized mice, exhibiting an improved tumor-killing efficacy. Mechanistically, CD70 knockout inhibits a collective lipid signaling network in CD4+ naïve and regulatory T cells involving mitochondrial integrity, cholesterol homeostasis, and fatty acid metabolism. Furthermore, ATAC-Seq delineates that CD70 is transcriptionally upregulated by NFKB2 via an Epstein-Barr virus-dependent epigenetic modification. Our findings identify CD70+ nasopharyngeal carcinoma cells as a metabolic switch that enforces the lipid-driven development, functional specialization and homeostasis of Tregs, leading to immune evasion. This study also demonstrates that CD70 blockade can act synergistically with anti-PD-1 treatment to reinvigorate T-cell immunity against nasopharyngeal carcinoma.

Environmental factors and gut microbiota: Toward better conservation of deer species.

Thousands of microbial species inhabiting the animal gut, collectively known as the gut microbiota, play many specific roles related to host nutrient metabolism and absorption, immune regulation, and protection from pathogenic bacteria. Gut microbiota composition is affected by several internal and external factors, such as the host genotype, dietary intake, breeding environment, and antibiotic exposure. As deer species are important members for maintaining ecosystem balance, understanding the effects of multiple factors on the gut microbiota of deer species, particularly endangered ones, is crucial. In this review, we summarize and discuss the factors that significantly affect the gut microbiota of deer and present the impacts of these factors on microbial composition. In particular, we focused on the changes in gut microbiota due to dietary differences under different conditions, including seasonal changes, different geographical locations, and captivity, as well as weaning and pathogen disturbance. Understanding the correlations between gut microbiota composition and its driving factors is important for evaluating and improving the captive breeding environment for better conservation of endangered deer species, and reintroducing wild deer populations in the future.

Estimation of cell lineages in tumors from spatial transcriptomics data.

Spatial transcriptomics (ST) technology through in situ capturing has enabled topographical gene expression profiling of tumor tissues. However, each capturing spot may contain diverse immune and malignant cells, with different cell densities across tissue regions. Cell type deconvolution in tumor ST data remains challenging for existing methods designed to decompose general ST or bulk tumor data. We develop the Spatial Cellular Estimator for Tumors (SpaCET) to infer cell identities from tumor ST data. SpaCET first estimates cancer cell abundance by integrating a gene pattern dictionary of copy number alterations and expression changes in common malignancies. A constrained regression model then calibrates local cell densities and determines immune and stromal cell lineage fractions. SpaCET provides higher accuracy than existing methods based on simulation and real ST data with matched double-blind histopathology annotations as ground truth. Further, coupling cell fractions with ligand-receptor coexpression analysis, SpaCET reveals how intercellular interactions at the tumor-immune interface promote cancer progression.

Classification and molecular characteristics of tet(X)-carrying plasmids in Acinetobacter species.

The rapid dissemination of plasmid-mediated tet(X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmid-mediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids.

A balanced gut microbiota is essential to maintain health in captive sika deer.

Certain animals harbor a high proportion of pathogens, particular the zoonotic pathogens, in their gut microbiome but are usually asymptomic; however, their carried pathogens may seriously threaten the public health. By understanding how the microbiome overcomes the negative effects of pathogens to maintain host health, we can develop novel solutions to control animal-mediated pathogen transmission including identification and application of beneficial microbes. Here, we analyzed the gut microbiota of 10 asymptomic captive sika deer individuals by full-length 16S rDNA sequencing. Twenty-nine known pathogens capable of infecting humans were identified, and the accumulated proportions of the identified pathogens were highly variable among individuals (2.33 to 39.94%). The relative abundances of several beneficial bacteria, including Lactobacillus and Bifidobacterium, were found to be positively correlated with the relative abundances of accumulated pathogens. Whole-genome metagenomic analysis revealed that the beneficial- and pathogenic-associated functions, such as genes involved in the synthesis of short chain fatty acids and virulence factors, were also positively correlated in the microbiome, indicating that the beneficial and pathogenic functions were maintained at a relatively balanced ratio. Furthermore, the bacteriophages that target the identified pathogens were found to be positively correlated with the pathogenic content in the microbiome. Several high-quality genomes of beneficial bacteria affiliated with Lactobacillus and Bifidobacterium and bacteriophages were recovered from the metagenomic data. Overall, this study provides novel insights into the interplay between beneficial and pathogenic content to ensure maintenance of a healthy gut microbiome, and also contributes to discovery of novel beneficial microbes and functions that control pathogens. KEY POINTS: • Certain asymptomic captive sika deer individuals harbor relatively high amounts of zoonotic pathogens. • The beneficial microbes and the beneficial functions are balanced with the pathogenic contents in the gut microbiome. • Several high-quality genomes of beneficial bacteria and bacteriophages are recovered by metagenomics.

CrisprVi: a software for visualizing and analyzing CRISPR sequences of prokaryotes.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and their spacers are important components of prokaryotic CRISPR-Cas systems. In order to analyze the CRISPR loci of multiple genomes more intuitively and comparatively, here we propose a visualization analysis tool named CrisprVi. RESULTS: CrisprVi is a Python package consisting of a graphic user interface (GUI) for visualization, a module for commands parsing and data transmission, local SQLite and BLAST databases for data storage and a functions layer for data processing. CrisprVi can not only visually present information of CRISPR direct repeats (DRs) and spacers, such as their orders on the genome, IDs, start and end coordinates, but also provide interactive operation for users to display, label and align the CRISPR sequences, which help researchers investigate the locations, orders and components of the CRISPR sequences in a global view. In comparison to other CRISPR visualization tools such as CRISPRviz and CRISPRStudio, CrisprVi not only improves the interactivity and effects of the visualization, but also provides basic statistics of the CRISPR sequences, and the consensus sequences of DRs/spacers across the input strains can be inspected from a clustering heatmap based on the BLAST results of the CRISPR sequences hitting against the genomes. CONCLUSIONS: CrisprVi is a convenient tool for visualizing and analyzing the CRISPR sequences and it would be helpful for users to inspect novel CRISPR-Cas systems of prokaryotes.

PICALM exerts a role in promoting CRC progression through ERK/MAPK signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumor in gastrointestinal tract with high incidence and mortality. In this study, the functions and potential mechanism of phosphatidylinositol-binding clathrin assembly protein (PICALM) in CRC were preliminarily explored. METHODS: Based on the Cancer Genome Atlas database and immunohistochemistry staining, revealing that the expression level of PICALM in CRC tissues was higher than that in adjacent normal tissues. RESULTS: Moreover, loss-of-function and gain-of-function assays in HCT 116 and RKO cells found that PICALM promotes proliferation and migration of CRC cells and inhibits apoptosis. Consistently, knockdown of PICALM inhibited tumorigenicity of CRC cells in vivo. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that knockdown of PICALM resulted in the enrichment of MAPK signaling pathway. Treatment of CRC cells with MAPK inhibitor reversed the effects of PICALM overexpression on proliferation and apoptosis. In addition, overexpression of PICALM upregulated the protein levels of ERK1/2 (p-ERK1/2), MEK1/2 (p-MEK1/2), p38 (p-p38) and JNK (p-JNK), and these effects were partially alleviated by the treatment of MAPK inhibitor. CONCLUSIONS: In summary, the study presented the new discovery that PICALM promoted CRC progression through ERK/MAPK signaling pathway, which drew further interest regarding its clinical application as a promising therapeutic target.

Campylobacter jejuni Developed the Resistance to Bacteriophage CP39 by Phase Variable Expression of 06875 Encoding the CGPTase.

Bacteriophage (phage) is regarded as an antimicrobial alternative for Campylobacter in food production. However, the development of phage resistance to the host is a main concern for the phage application. This study characterized the phage CP39 and investigated the phage resistance of CP39 in Campylobacter jejuni NCTC12662. We determined that phage CP39 belonged to the Myoviridae family by the WGS and phylogenetic analysis. Phage CP39 was confirmed as a capsular polysaccharide (CPS)-dependent phage by primary C. jejuni phage typing. It was further confirmed that the phage could not be adsorbed by the acapsular mutant ΔkpsM but showed the same lytic ability in both the wild-type strain NCTC 12662 and the ΔmotA mutant lacking motile flagella filaments. We further determined that the 06875 gene encoding CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase (CGPTase) in the CPS loci was related to phage CP39 adsorption by SNP analysis and observed a rapid development of phage resistance in NCTC 12662 during the phage infection. Furthermore, we observed a high mutation frequency of 06875 (32%), which randomly occurred in nine different sites in the gene according to colony PCR sequencing. The mutation of the 06875 gene could cause the phase variable expression of non-functional protein and allow the bacteria against the phage infection by modifying the CPS. Our study confirmed the 06875 gene responsible for the CPS-phage adsorption for the first time and demonstrated the phase variable expression as a main mechanism for the bacteria to defend phage CP39. Our study provided knowledge for the evolutionary adaption of bacteria against the bacteriophage, which could add more information to understand the phage resistance mechanism before applying in the industry.

Prevalence and characteristics of Campylobacter from the genital tract of primates and ruminants in Eastern China.

Campylobacter infection is an important cause of genital failure in ruminants in developed countries. Although historically Campylobacter fetus subspecies fetus has been the main cause of abortion in sheep, C. jejuni is also increasingly associated with sheep abortions. However, limited information is known on Campylobacter-associated abortions in China. This study initially investigated the distribution of Campylobacter from the genital tracts of humans, monkeys, sheep and cows in China from 2017 to 2018. Ten out of 2126 (0.47%) samples from the genital tracts were Campylobacter positive, of which seven (70%) isolates were identified as C. jejuni. Phylogenetic analysis showed the high genetic diversity of these isolates. The human isolates were closely related to the sheep isolates implying inter-transmission of Campylobacter between humans and sheep according to the phylogenetic analysis. The acid resistance, adhesion and invasion abilities of genital tract isolates were stronger than isolates from gastrointestinal tract, but no significant difference was observed in the virulence genes. We further found that three genital tract isolates belonged to the same cluster as gastrointestinal isolates from the same host. These findings suggested that there may be inter-transmission of Campylobacter between the genital and gastrointestinal tract.

Investigating the role of BN-domains of FlhF involved in flagellar synthesis in Campylobacter jejuni.

FlhF protein is critical for intact flagellar assembly in Campylobacter jejuni. It is a putative GTPase with B-, N- and G-domains. However, the role of the B- and N-domains in flagella biosynthesis remains unclear in C. jejuni. This study demonstrated that both the B- and N-domains are essential for flagellar synthesis, with the absence of B- and/or N-domains showing truncated variants of FlhF by TEM. Point mutations in the B- and N-domains (T13A, K159A, G231A) also induced flagella abnormalities. Furthermore, significant defects in GTPase activity and polar targeting of FlhF were triggered by point mutations of B- and N-domains. Flagella gene expression and transcription were also significantly disrupted in flhF(T13A), flhF(K159A) and flhF(G231A) strains. This study initially explored the effects of B- and N-domains on flagella synthesis. We speculated that B- and N-domains may directly or indirectly cause flagella abnormalities by affecting flagellar gene expression or GTPase activity, which helps us better understand the function of FlhF in flagella synthesis.