vB-ApyS-JF1, the First Trueperella pyogenes Phage, Shows Potential as an Alternative Treatment Strategy for Trueperella pyogenes Infections.

Trueperella pyogenes (T. pyogenes) is an important opportunistic animal pathogen that causes huge economic losses to the animal husbandry industry. The emergence of bacterial resistance and the unsatisfactory effect of the vaccine have prompted investigators to explore alternative strategies for controlling T. pyogenes infection. Due to the ability of phages to kill multidrug-resistant bacteria, the use of phage therapy to combat multidrug-resistant bacterial infections has attracted attention. In this study, a T. pyogenes phage, vB-ApyS-JF1 (JF1), was isolated from sewage samples, and its whole genome and biological characteristics were elucidated. Moreover, the protective effect of phage JF1 on a mouse bacteremic model caused by T. pyogenes was studied. JF1 harbors a double-stranded DNA genome with a length of 90,130 bp (30.57% G + C). The genome of JF1 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. Moreover, the genome sequence of JF1 exhibited low coverage (<6%) with all published phages in the NCBI database, and a phylogenetic analysis of the terminase large subunits and capsid indicated that JF1 was evolutionarily distinct from known phages. In addition, JF1 was stable over a wide range of pH values (3 to 11) and temperatures (4 to 50°C) and exhibited strong lytic activity against T. pyogenes in vitro. In murine experiments, a single intraperitoneal administration of JF1 30 min post-inoculation provided 100% protection for mice against T. pyogenes infection. Compared to the phosphate-buffered saline (PBS) treatment group, JF1 significantly (P < 0.01) reduced the bacterial load in the blood and tissues of infected mice. Meanwhile, treatment with phage JF1 relieved the pathological symptoms observed in each tissue. Furthermore, the levels of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in the blood of infected mice were significantly (P < 0.01) decreased in the phage-treated group. Taken together, these results indicate that phage JF1 demonstrated great potential as an alternative therapeutic treatment against T. pyogenes infection.

TRIM21 negatively regulates Corynebacterium pseudotuberculosis-induced inflammation and is critical for the survival of C. pseudotuberculosis infected C57BL6 mice.

Corynebacterium pseudotuberculosis, a facultative intracellular bacterium, is an important zoonotic pathogen responsible for chronic inflammatory diseases. TRIM21, an E3 ubiquitin-protein ligase, plays pivotal roles in inflammation regulation. However, its role during C. pseudotuberculosis infection is unclear. Here, we found that TRIM21 expression was significantly increased in C. pseudotuberculosis-infected macrophages. Following infection by C. pseudotuberculosis, we observed a significantly higher number of bacteria and a higher degree of LDH release from Trim21-/- macrophages compared to wild-type (WT) macrophages, suggesting that TRIM21 limits C. pseudotuberculosis replication in macrophages and protects the infected cells from death. Further in vivo experiments showed a significantly higher mortality, higher bacterial load, much more severe abscess formation, and lesions in the organs of C. pseudotuberculosis-infected Trim21-/- mice compared to those of the infected WT mice, suggesting that TRIM21 plays critical roles in protecting against C. pseudotuberculosis infection. Moreover, the secretory levels of IL-1α, IL-1ß, IL-6, and TNF-α were significantly higher in C. pseudotuberculosis-infected Trim21-/- macrophages compared to infected WT macrophages; the levels of these cytokines were also higher in the sera, organs, and ascites of C. pseudotuberculosis-infected Trim21-/- mice compared to infected WT mice. These findings suggest that TRIM21 negatively regulates the secretion of pro-inflammatory cytokines in macrophages, sera, organs, and ascites of mice following C. pseudotuberculosis infection. Collectively, the present study demonstrates that TRIM21 plays a vital role in preventing C. pseudotuberculosis infection, which may be related to the negative regulation of pro-inflammatory cytokines production by TRIM21 during this pathogen infection.

Cofilin-1, peroxiredoxin-1, and galectin-3: Major proteins released by macrophages infected with Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a broad host-spectrum zoonotic pathogen, causes caseous lymphadenitis (CLA) in small ruminants and is responsible for considerable economic losses in the livestock industry worldwide. Macrophages play a pivotal role in the immunopathogenesis of CLA. However, the immunoregulatory mechanisms of macrophages against C. pseudotuberculosis remains poorly understood. In the present study, for the first time, the partial exoproteome of murine peritoneal macrophages infected with C. pseudotuberculosis was profiled and the differential expression of the identified proteins was analyzed. In macrophages, infection with C. pseudotuberculosis, rather than with heat-killed bacteria, induced release of diverse proteins. Three unconventional proteins: cofilin-1, peroxiredoxin-1, and galectin-3 were significantly expressed and released by infected macrophages into the culture supernatant. These proteins are involved in the host inflammatory response and may be responsible for the excessive inflammation of CLA. In C. pseudotuberculosis-infected macrophages, the release of cofilin-1 and peroxiredoxin-1 was predominant at later stages of infection, while the release of galectin-3 was independent of time. Taken together, the present work contributes to our understanding of the functional role of macrophage response to C. pseudotuberculosis infection.

Critical roles of NLRP3 inflammasome in IL-1ß secretion induced by Corynebacterium pseudotuberculosis in vitro.

Corynebacterium pseudotuberculosis is a prominent human and animal pathogen causing chronic inflammatory diseases. Interleukin-1ß (IL-1ß) is involved in the response to such pathogenic infections. However, the mechanism by which IL-1ß is secreted during C. pseudotuberculosis infection remains unclear. This study aimed to investigate the mechanism underlying IL-1ß secretion by macrophages infected with C. pseudotuberculosis. Herein, we firstly revealed that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Casp1) play critical roles in IL-1ß secretion rather than IL-1ß precursor (pro-IL-1ß) expression in C. pseudotuberculosis-infected macrophages. Toll like receptor 4 (TLR4) is partially involved in IL-1ß secretion, while absent in melanoma 2 (AIM2) is not involved in IL-1ß secretion by C. pseudotuberculosis-infected macrophages. In addition, nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinases (p38 MAPK) inhibitors almost attenuated IL-1ß secretion, implying that NF-κB and p38MAPK pathway are involved in IL-1ß secretion in C. pseudotuberculosis-infected macrophages. Furthermore, C. pseudotuberculosis were significantly more numerous in Nlrp3-/-, Asc-/-, and Casp-1-/- macrophages than in WT macrophages at 24 h after infection (P < 0.05), indicating that NLRP3 inflammasome components limit C. pseudotuberculosis replication in macrophages. Together, these data provide novel insights into the mechanisms underlying IL-1ß secretion in C. pseudotuberculosis-infected macrophages and further the current understanding of the host pro-inflammatory immune response against this pathogen.

Sodium butyrate ameliorates Corynebacterium pseudotuberculosis infection in RAW264.7 macrophages and C57BL/6 mice.

Corynebacterium pseudotuberculosis (CP) infection in livestock has become highly difficult to control. To decrease the incidence of CP infection, the supplementation of feed with non-antibiotic antibacterial substances is a potential approach. The aim of this study was to assess the effects of sodium butyrate (NaB), a potential alternative to antibiotics, on CP infection in RAW264.7 macrophages and C57BL/6 mice. Our data showed that NaB (2 mM) significantly ameliorated CPinfection in RAW264.7 macrophages and decreased the bacterial load in the spleens of infected mice. By real-time PCR, we found that NaB induced significant decreases in zinc-dependent superoxide dismutase (sodC) and tip protein C (spaC) expression in CP from infected-RAW264.7 cells and in phospholipase D (pld) and spaC expression in CP from the spleens of infected mice. NaB treatment significantly up-regulated cathelicidin-related antimicrobial peptide (cramp) expression in spleens of mice infected with CP. Furthermore, NaB alleviated histopathological changes in spleens of CP-infected mice. In conclusion, NaB ameliorated CP infection in RAW264.7 macrophages and C57BL/6 mice, and these effects may be related to the modulation of sodC, spaC, pld, and cramp expression.

Characterization of Klebsiella pneumoniae associated with cattle infections in southwest China using multi-locus sequence typing (MLST), antibiotic resistance and virulence-associated gene profile analysis

Abstract Klebsiella pneumoniae is important human and animal pathogen that causes a wide spectrum of infections. In this study, isolates from cattle nasal swabs samples were identified by 16S rRNA, and to evaluate the antimicrobial susceptibility, virulence gene carrying levels, and multilocus sequence typing of K. pneumoniae isolates. 33 isolates of K. pneumoniae were isolated and identified in 213 nasal swabs samples, of which 12 were hypervirulent K. pneumoniae strains. Extended Spectrum Beta-Lactamases genes were found in 93.4% of the strains. Of which, TEM was the most prevalent (93.4%), followed by CTX-M and SHV were 57.6% and 39.4%, respectively. A main mutation pattern of quinoloneresistance-determining region, Thr83-Ieu and Asp87-Asn in gyrA and Ser87-Ile in parC, was detected in 33 K. pneumoniae isolates. All the isolates harbored at least two virulence factor genes, with ureA (97.0%) and wabG (91.0%) exhibiting high carriage rates in 33 K. pneumoniae isolates. MLST revealed 7 sequence types, of which 3 STs (2541, 2581 and 2844) were newly assigned. Using eBURST, ST2844 and ST2541 were assigned to new clonal complex 2844. Our study provides evidence and biological characteristics of K. pneumoniae isolates from cattle upper respiratory tract in Southwest China.

Characterization of Klebsiella pneumoniae associated with cattle infections in southwest China using multi-locus sequence typing (MLST), antibiotic resistance and virulence-associated gene profile analysis

Abstract Klebsiella pneumoniae is important human and animal pathogen that causes a wide spectrum of infections. In this study, isolates from cattle nasal swabs samples were identified by 16S rRNA, and to evaluate the antimicrobial susceptibility, virulence gene carrying levels, and multilocus sequence typing of K. pneumoniae isolates. 33 isolates of K. pneumoniae were isolated and identified in 213 nasal swabs samples, of which 12 were hypervirulent K. pneumoniae strains. Extended Spectrum Beta-Lactamases genes were found in 93.4% of the strains. Of which, TEM was the most prevalent (93.4%), followed by CTX-M and SHV were 57.6% and 39.4%, respectively. A main mutation pattern of quinoloneresistance-determining region, Thr83-Ieu and Asp87-Asn in gyrA and Ser87-Ile in parC, was detected in 33 K. pneumoniae isolates. All the isolates harbored at least two virulence factor genes, with ureA (97.0%) and wabG (91.0%) exhibiting high carriage rates in 33 K. pneumoniae isolates. MLST revealed 7 sequence types, of which 3 STs (2541, 2581 and 2844) were newly assigned. Using eBURST, ST2844 and ST2541 were assigned to new clonal complex 2844. Our study provides evidence and biological characteristics of K. pneumoniae isolates from cattle upper respiratory tract in Southwest China.

Upregulation of chicken TLR4, TLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with Eimeria tenella in vitro.

Coccidiosis, caused by Eimeria parasites, is a major parasitic disease responsible for great economic losses in the poultry industry. Toll-like receptor (TLR) family is one of the most important innate immune receptors, which involved in pathogen detection by initiating host responses, and it plays important roles in the reduction and clearance of pathogens. Very little information is available about the roles of chicken TLRs (ChTLRs) during Eimeria tenella infection. In the current study, mRNA expression of ChTLRs and associated signal adaptors in heterophils and monocyte-derived macrophages stimulated with E. tenella in vitro were measured by real-time quantitative polymerase chain reaction. The results showed that ChTLR4 and ChTLR15 expression were increased significantly in heterophils and monocyte-derived macrophages following live E. tenella sporozoites stimulation. The heat-killed E. tenella sporozoites stimulated higher expression of ChTLRs and signal adaptors than live sporozoites, the expression of ChTLR4, ChTLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with heat-killed E. tenella sporozoites were up-regulated significantly than unstimulated cells. The results suggest that ChTLR4 and ChTLR15 are involved in response to E. tenella infection, and may operate in a MyD88-dependent manner for host defense.