Pasteurella multocida activates apoptosis via the FAK-AKT-FOXO1 axis to cause pulmonary integrity loss, bacteremia, and eventually a cytokine storm.

Pasteurella multocida is an important zoonotic respiratory pathogen capable of infecting a diverse range of hosts, including humans, farm animals, and wild animals. However, the precise mechanisms by which P. multocida compromises the pulmonary integrity of mammals and subsequently induces systemic infection remain largely unexplored. In this study, based on mouse and rabbit models, we found that P. multocida causes not only lung damage but also bacteremia due to the loss of lung integrity. Furthermore, we demonstrated that bacteremia is an important aspect of P. multocida pathogenesis, as evidenced by the observed multiorgan damage and systemic inflammation, and ultimately found that this systemic infection leads to a cytokine storm that can be mitigated by IL-6-neutralizing antibodies. As a result, we divided the pathogenesis of P. multocida into two phases: the pulmonary infection phase and the systemic infection phase. Based on unbiased RNA-seq data, we discovered that P. multocida-induced apoptosis leads to the loss of pulmonary epithelial integrity. These findings have been validated in both TC-1 murine lung epithelial cells and the lungs of model mice. Conversely, the administration of Ac-DEVD-CHO, an apoptosis inhibitor, effectively restored pulmonary epithelial integrity, significantly mitigated lung damage, inhibited bacteremia, attenuated the cytokine storm, and reduced mortality in mouse models. At the molecular level, we demonstrated that the FAK-AKT-FOXO1 axis is involved in P. multocida-induced lung epithelial cell apoptosis in both cells and animals. Thus, our research provides crucial information with regard to the pathogenesis of P. multocida as well as potential treatment options for this and other respiratory bacterial diseases.

Pasteurella multocida activates Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis.

Pasteurella multocida is an opportunistic zoonotic pathogen that primarily causes fatal respiratory diseases, such as pneumonia and respiratory syndromes. However, the precise mechanistic understanding of how P. multocida disrupts the epithelial barrier in mammalian lung remains largely unknown. In this study, using unbiased RNA-seq analysis, we found that the evolutionarily conserved Hippo-Yap pathway was dysregulated after P. multocida infection. Given the complexity of P. multocida infection associated with lung injury and systemic inflammatory processes, we employed a combination of cell culture models, mouse models, and rabbit models to investigate the dynamics of the Hippo-Yap pathway during P. multocida infection. Our findings reveal that P. multocida infection activates the Hippo-Yap pathway both in vitro and in vivo, by upregulating the upstream factors p-Mst1/2, p-Lats1, and p-Yap, and downregulating the downstream effectors Birc5, Cyr61, and Slug. Conversely, pharmacological inhibition of the Hippo pathway by XMU-MP-1 significantly rescued pulmonary epithelial cell apoptosis in vitro and reduced lung injury, systemic inflammation, and mouse mortality in vivo. Mechanistic studies revealed that P. multocida induced up-regulation of Rassf1 expression, and Rassf1 enhanced Hippo-Yap pathway through phosphorylation. Accordingly, in vitro knockdown of Rassf1 significantly enhanced Yap activity and expression of Yap downstream factors and reduced apoptosis during P. multocida infection. P. multocida-infected rabbit samples also showed overexpression of Rassf1, p-Lats1, and p-Yap, suggesting that P. multocida activates the Rassf1-Hippo-Yap pathway. These results elucidate the pathogenic role of the Rassf1-Hippo-Yap pathway in P. multocida infection and suggest that this pathway has the potential to be a drug target for the treatment of pasteurellosis.

Attenuated vaccine PmCQ2Δ4555-4580 effectively protects mice against Pasteurella multocida infection.

Pasteurella multocida type A (PmA) mainly causes respiratory diseases such as pneumonia in bovines, leading to great economic losses to the breeding industry. At present, there is still no effective commercial vaccine against PmA infection. In this study, a mutant strain (PmCQ2Δ4555-4580) with brand-new phenotypes was obtained after serially passaging at 42 °C. Whole genome resequencing and PCR analysis showed that PmCQ2Δ4555-4580 missed six genes, including PmCQ2_004555, PmCQ2_004560, PmCQ2_004565, PmCQ2_004570, PmCQ2_004575, and PmCQ2_004580. Importantly, the virulence of PmCQ2Δ4555-4580 was reduced by approximately 2.8 × 109 times in mice. Notably, live PmCQ2Δ4555-4580 could provide 100%, 100% and 40% protection against PmA, PmB and PmF, respectively; and inactivated PmCQ2Δ4555-4580 could provide 100% and 87.5% protection against PmA and PmB. Interestingly, immune protection-related proteins were significantly upregulated in PmCQ2Δ4555-4580 based on RNA-seq and bioinformatics analysis. Meaningfully, by in vitro expression, purification and in vivo immunization, 12 proteins had different degrees of immune protective effects. Among them, PmCQ2_008205, PmCQ2_010435, PmCQ2_008190, and PmCQ2_004170 had the best protective effect, the protection rates against PmA were 50%, 40%, 30%, and 30%, respectively, and the protective rates against PmB were 62.5%, 42.9%, 37.5%, and 28.6%, respectively. Collectively, PmCQ2Δ4555-4580 is a potential vaccine candidate for the prevention of Pasteurellosis involving in high expression of immune protective related proteins.

Isolation and Genomic Characterization of a Chinese Genotype C Bovine Parainfluenza Virus Type 3 from Cattle and Its Pathogenicity in C57BL/6 Mice.

Bovine parainfluenza virus type 3 (BPIV-3), also known as bovine respirovirus 3, is a common respiratory pathogen associated with bovine respiratory disease (BRD). BPIV-3 has currently circulated worldwide; however, data on the prevalence and genetic characteristics of BPIV-3 are still scarce and limited. In this study, the BPIV-3 strain SC was identified and isolated from cattle presenting with clinical signs of BRD in China. Animal experiments indicated that BPIV-3 SC can successfully infect C57BL/6 mice and induce weight loss, lung inflammatory cell infiltration, and inflammatory cytokine expression in mice. In addition, the complete genome of BPIV-3 SC was obtained using next-generation sequencing and was 15,473 bp in length. Phylogenetic analysis indicated that BPIV-3 SC belonged to genotype C, which clustered in the same large clade consisting of a population of Chinese genotype C strains but was found to be different from the other strains upon further differentiation. Compared to other Chinese genotype C strains, the BPIV-3 SC showed 70 unique nucleotide mutations and 13 unique amino acid mutations in the HN, P, and L proteins, suggesting a unique genetic evolution of BPIV-3 SC. In conclusion, we isolated and characterized a differential Chinese genotype C BPIV-3, which contributed to an understanding of the prevalence and evolution of BPIV-3 in China.

Secretome Analysis of High- and Low-Virulent Bovine Pasteurella multocida Cultured in Different Media.

Bovine Pasteurella multocida (P. multocida) serotype A is one of the major causes of bovine respiratory disease (BRD). We used data-independent acquisition (DIA) LC-MS/MS combined with bioinformatics analysis to identify proteins secreted by P. multocida. A total of 154 proteins were obtained from the supernatants of two isolates of bovine P. multocida serotype A (high virulent PmCQ2 and low virulent PmCQ6) cultured in Martin or BHI media, of which 50 were identified as putative secreted proteins. Further studies showed that Tuf, an elongation factor Tu, was highly expressed in P. multocida and secreted into infected tissues. Tuf stimulated strong innate immune responses of macrophages and had protective efficacy against P. multocida infection in a mouse model. The results provide insight into the secreted proteins of P. multocida and suggest new targets for vaccine development against P. multocida.

Corrigendum: L-ascorbic acid shapes bovine Pasteurella multocida serogroup A infection.

[This corrects the article DOI: 10.3389/fvets.2021.687922.].

Emergence and genomic analysis of a novel sublineage of bovine ephemeral fever virus in Southwest China.

Introduction: Bovine ephemeral fever virus (BEFV), belonging to the genus Ephemerovirus under the family Rhabdoviridae, is the etiological cause for the bovine ephemeral fever (BEF) in cattle and water buffalo. Methods: In this study, we report recent BEF outbreaks in Southwest China and sequence the complete genome sequence of one BEFV isolate BEFV/CQ1/2022. Results and Discussion: Comparative genomic analyses between BEFV/CQ1/2022 and isolates available in GenBank revealed remarkable inter-isolate divergence. Meanwhile, the sequence divergence was related to the evolutionary relationships and geographical distribution of the isolates. Phylogenetic analysis indicated that the global BEFV isolates can be divided into 4 distinct lineages. The East Asia lineage was the most diverse and could be subdivided into 4 sublineages. Notably, BEFV/CQ1/2022 and other 10 recent isolates from Mainland China were found to be clustered in sublineage 2. Additionally, recombination analysis provided evidence of BEFV recombination among East Asian isolates for the first time. Taken together, a novel sublineage of the East Asian BEFV emerged in Southwest China, and large divergence and potential recombination among BEFV strains were investigated in this study, which may improve understanding of BEFV epidemiology and evolution.

Slc6a13 Deficiency Attenuates Pasteurella multocida Infection-Induced Inflammation via Glycine-Inflammasome Signaling.

We have previously demonstrated that Slc6a13-deficient (Slc6a13-/-; KO) mice are resistant to P. multocida infection, which might be in connection with macrophage-mediated inflammation; however, the specific metabolic mechanism is still enigmatic. Here we reproduce the less sensitive to P. multocida infection in overall survival assays as well as reduced bacterial loads, tissue lesions, and inflammation of lungs in KO mice. The transcriptome sequencing analysis of wild-type (WT) and KO mice shows a large number of differentially expressed genes that are enriched in amino acid metabolism by functional analysis. Of note, glycine levels are substantially increased in the lungs of KO mice with or without P. multocida infection in comparison to the WT controls. Interestingly, exogenous glycine supplementation alleviates P. multocida infection-induced inflammation. Mechanistically, glycine reduces the production of inflammatory cytokines in macrophages by blocking the activation of inflammasome (NALP1, NLRP3, NLRC4, AIM2, and Caspase-1). Together, Slc6a13 deficiency attenuates P. multocida infection through lessening the excessive inflammatory responses of macrophages involving glycine-inflammasome signaling.

Melatonin inhibits Gram-negative pathogens by targeting citrate synthase.

Bacterial infections caused by Gram-negative pathogens represent a growing burden for public health worldwide. Despite the urgent need for new antibiotics that effectively fight against pathogenic bacteria, very few compounds are currently under development or approved in the clinical setting. Repurposing compounds for other uses offers a productive strategy for the development of new antibiotics. Here we report that the multifaceted melatonin effectively improves survival rates of mice and decreases bacterial loads in the lung during infection. Mechanistically, melatonin specifically inhibits the activity of citrate synthase of Gram-negative pathogens through directly binding to the R300, D363, and H265 sites, particularly for the notorious Pasteurella multocida. These findings highlight that usage of melatonin is a feasible and alternative therapy to tackle the increasing threat of Gram-negative pathogen infections via disrupting metabolic flux of bacteria.

Deleting qseC downregulates virulence and promotes cross-protection in Pasteurella multocida.

QseC, a histidine sensor kinase of the QseBC two-component system, acts as a global regulator of bacterial stress resistance, biofilm formation, and virulence. The function of QseC in some bacteria is well understood, but not in Pasteurella multocida. We found that deleting qseC in P. multocida serotype A:L3 significantly down-regulated bacterial virulence. The mutant had significantly reduced capsule production but increased resistance to oxidative stress and osmotic pressure. Deleting qseC led to a significant increase in qseB expression. Transcriptome sequencing analysis showed that 1245 genes were regulated by qseC, primarily those genes involved in capsule and LPS biosynthesis and export, biofilm formation, and iron uptake/utilization, as well as several immuno-protection related genes including ompA, ptfA, plpB, vacJ, and sodA. In addition to presenting strong immune protection against P. multocida serotypes A:L1 and A:L3 infection, live ΔqseC also exhibited protection against P. multocida serotype B:L2 and serotype F:L3 infection in a mouse model. The results indicate that QseC regulates capsular production and virulence in P. multocida. Furthermore, the qseC mutant can be used as an attenuated vaccine against P. multocida strains of multiple serotypes.

A single point mutation in the hyaC gene affects Pasteurella multocida serovar A capsule production and virulence.

Pasteurella multocida (P. multocida) is a Gram-negative bacterium which causes diseases in poultry, livestock, and humans, resulting in huge economic losses. P. multocida serovar A CQ6 (PmCQ6) is a naturally occurring attenuated strain with a thin capsule. Thus, we aimed to explore why this strain is less virulent and produces less capsule compared with P. multocida serovar A strain CQ2 (PmCQ2). Analysis of capsular polysaccharide synthesis genes in PmCQ6 revealed that, compared with PmCQ2, there was only a single point mutation in the initiation codon sequence of the hyaC gene. To test whether this point mutation caused capsular deficiency and reduced virulence, we rescued this hyaC mutation and observed a restoration of capsule production and higher virulence. Transcriptome analysis showed that the hyaC point mutation led to a downregulation of capsule synthesis and/or iron utilization related-genes. Taken together, the results indicate that the start codon mutation of hyaC is an important factor affecting the capsule synthesis and virulence of PmCQ6.

L-Ascorbic Acid Shapes Bovine Pasteurella multocida Serogroup A Infection.

Bovine Pasteurella multocida serogroup A (bovine PmA) is one of the most important pathogens causing fatal pneumonia in cattle. However, it is largely unknown how nutrition shapes bovine PmA infection. Here, we discovered that the infected lung held the highest bacterial density than other tissues during infection. By screening the different metabolites between high (lung)- and low (liver)-bacterial density tissues, the present work revealed that L-ascorbic acid and L-aspartic acid directly influenced bovine P. multocida growth. Interestingly, L-ascorbic acid, which is expressed at higher levels in the infected livers, inhibited bovine PmA growth as well as virulence factor expression and promoted macrophage bactericidal activity in vitro. In addition, ascorbic acid synthesis was repressed upon bovine PmA infection, and supplementation with exogenous L-ascorbic acid significantly reduced the bacterial burden of the infected lungs and mouse mortality. Collectively, our study has profiled the metabolite difference of the murine lung and liver during bovine PmA infection. The screened L-ascorbic acid showed repression of bovine PmA growth and virulence expression in vitro and supplementation could significantly increase the survival rate of mice and reduce the bacterial load in vivo, which implied that L-ascorbic acid could serve as a potential protective agent for bovine PmA infection in clinic.

Transcriptomic Analysis of High- and Low-Virulence Bovine Pasteurella multocida in vitro and in vivo.

Pasteurella multocida is a gram-negative opportunistic pathogen that causes various diseases in poultry, livestock, and humans, resulting in huge economic losses. Pasteurella multocida serotype A CQ6 (PmCQ6) is a naturally occurring attenuated strain, while P. multocida serotype A strain CQ2 (PmCQ2) is a highly virulent strain isolated from calves. Compared with PmCQ2, it was found that bacterial loads and tissue lesions of lung tissue significantly decreased and survival rates significantly improved in mice infected with PmCQ6 by intranasal infection. However, comparative genome analysis showed that the similarity between the two strains is more than 99%. To further explore the virulence difference mechanism of PmCQ2 and PmCQ6, transcriptome sequencing analysis of the two strains was performed. The RNA sequencing analysis of PmCQ2 and PmCQ6 showed a large number of virulence-related differentially expressed genes (DEGs) in vivo and in vitro. Among them, 38 virulence-related DGEs were significantly up-regulated due to PmCQ6 infection, while the number of PmCQ2 infection was 46, much more than PmCQ6. In addition, 18 virulence-related DEGs (capsule, iron utilization, lipopolysaccharide, and outer membrane protein-related genes) were up-regulated in PmCQ2 infection compared to PmCQ6 infection, exhibiting a higher intensive expression level in vivo. Our findings indicate that these virulence-related DEGs (especially capsule) might be responsible for the virulence of PmCQ2 and PmCQ6, providing prospective candidates for further studies on pathogenesis.

The Role of Innate Immunity in Pulmonary Infections.

Innate immunity forms a protective line of defense in the early stages of pulmonary infection. The primary cellular players of the innate immunity against respiratory infections are alveolar macrophages (AMs), dendritic cells (DCs), neutrophils, natural killer (NK) cells, and innate lymphoid cells (ILCs). They recognize conserved structures of microorganisms through membrane-bound and intracellular receptors to initiate appropriate responses. In this review, we focus on the prominent roles of innate immune cells and summarize transmembrane and cytosolic pattern recognition receptor (PRR) signaling recognition mechanisms during pulmonary microbial infections. Understanding the mechanisms of PRR signal recognition during pulmonary pathogen infections will help us to understand pulmonary immunopathology and lay a foundation for the development of effective therapies to treat and/or prevent pulmonary infections.

Pasteurella multocida Pm0442 Affects Virulence Gene Expression and Targets TLR2 to Induce Inflammatory Responses.

Pasteurella multocida is an important pathogenic bacterium of domestic animals. However, the mechanisms of infection are still poorly understood. Here, we found that Pm0442 was dramatically up-regulated in infected mice among 67 predicted lipoproteins of P. multocida serotype A CQ2 strain (PmCQ2). To explore the role of Pm0442 in virulence and the potential of the mutant as a vaccine, Pm0442 mutant of PmCQ2 was successfully constructed. Then, the virulence characteristics, immune/inflammatory responses, and the survival rates of challenged mice were determined. As a result, it was found that the Pm0442 deletion of PmCQ2 significantly decreased bacterial loads and inflammatory responses of lung tissue in mice, resulting in improved survival. Mechanically, Pm0442 affects PmCQ2 capsular and lipopolysaccharide (LPS) synthesis and iron utilization-related genes expression affecting adhesion and phagocytosis. Furthermore, PM0442 bound directly to Toll-like receptor 2 (TLR2) to mediate the secretion of pro-inflammatory cytokine (IL-1ß, TNF-α, IL-6, and IL-12p40) in macrophages via activation of the NF-κB, ERK1/2 and p38 signaling pathways. Notably, PmCQ2Δ0442 could provide 70-80% protection to mice challenged with 3.08 × 107 CFU of PmCQ2. Our findings demonstrate that Pm0442 is a virulence-related gene of PmCQ2, which provides new guidance for the prevention and control of Pasteurellosis.

Transcriptomic Analysis of Chicken Lungs Infected With Avian and Bovine Pasteurella multocida Serotype A.

Pasteurella multocida (P. multocida) is a common animal pathogen responsible for many animal diseases. Strains from different hosts exhibit disparate degrees of effect in other species. Here, we characterize an avian P. multocida serogroup A strain (PmQ) showing high lethality to chickens and a bovine P. multocida serogroup A strain (PmCQ2) with no lethality to chickens. We used RNA-seq to profile the transcriptomes of chicken lungs infected with PmQ and PmCQ2. A total of 1,649 differentially expressed genes (DEGs) due to PmQ infection (831 upregulated genes and 818 downregulated genes) and 1427 DEGs (633 upregulated genes and 794 downregulated genes) due to PmCQ2 infection were identified. Functional analysis of these DEGs demonstrated that the TNF signaling pathway, the toll-like receptor signaling pathway, complement and coagulation cascades, and cytokine-cytokine receptor interaction were both enriched in PmQ and PmCQ2 infection. STAT and apoptosis signaling pathways were uniquely enriched by PmQ infection, and the NOD-like receptor signaling pathway was enriched only by PmCQ2 infection. Cell-type enrichment analysis of the transcriptomes showed that immune cells, including macrophages and granulocytes, were enriched in both infection groups. Collectively, our study profiled the transcriptomic response of chicken lungs infected with P. multocida and provided valuable information to understand the chicken responses to P. multocida infection.

High- and low-virulent bovine Pasteurella multocida induced differential NLRP3 inflammasome activation and subsequent IL-1ß secretion.

Pasteurella multocida is a gram-negative bacterial pathogen, which causes a large number of diseases in mammals, birds and human. Although the bacterium has been known for decades, the pathogenesis and the mechanisms of P. multocida induced host immunity are poorly understood. Recently, we have reported that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome plays an important role in caspase-1 activation and IL-1ß secretion in macrophages infected with P. multocida. In this study, the inflammasome activation and IL-1ß secretion were further demonstrated by using high- and low-virulent bovine P. multocida isolates. The results showed that, comparing with macrophages infected with the high-virulent PmCQ2 isolates, the low-virulent PmCQ6 induced higher levels of NLRP3 transcription, caspase-1 activation and mature IL-1ß secretion. Furthermore, the capsule of the high-virulent PmCQ2 was much thicker than that of low-virulent PmCQ6, which indicating that capsular thickness might influence the bacteria colonization and NLRP3 inflammasome activation. The results suggested that differences in maturation of IL-1ß in macrophages upon high- and low- virulent P. multocida infection are critically dependent on the differential activation of NLRP3 inflammasome. This study provided more understanding for the host immune responses induced by P. multocida and further extended the knowledge of P. multocida virulence from the view of host innate immunity.

l-Serine Lowers the Inflammatory Responses during Pasteurella multocida Infection.

Pasteurella multocida causes a variety of infectious diseases in various species of mammals and birds, resulting in enormous economic loss to the modern livestock and poultry industry. However, the mechanism of host-pathogen interaction is unclear. Here, we found that l-serine levels were significantly decreased in murine lungs infected with P. multocida Exogenous l-serine supplementation significantly increased the survival rate of mice and decreased the colonization of P. multocida in the lungs of mice. Notably, l-serine decreased the macrophage- and neutrophil-mediated inflammatory responses in mice during P. multocida infection.

NLRP3 inflammasome plays an important role in caspase-1 activation and IL-1ß secretion in macrophages infected with Pasteurella multocida.

Pasteurella multocida is a Gram-negative bacterium that is responsible for a variety of diseases in birds and mammals, including humans. We have previously reported that the P. multocida serotype A strain PmCQ2 causes severe lung pneumonia in bovines. Transcriptomic analysis showed that many genes related to the immune response were significantly upregulated in the lungs of mice infected with P. multocida compared with uninfected mice. However, the mechanism by which P. multocida induces host inflammatory cytokine secretion is poorly understood. In this study, the mechanism of caspase-1 activation and subsequent IL-1ß secretion in macrophages infected with P. multocida was elucidated. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome was shown to be involved in inducing this cellular response. Compared with wild-type macrophages, Nlrp3-/- macrophages exhibited a clear decrease in caspase-1 activation and IL-1ß secretion in response to P. multocida infection. Furthermore, spleen tyrosine kinase (Syk) was indicated to be involved in IL-1ß secretion, possibly by regulating the NLRP3 inflammasome. Our results provide new insight into the host proinflammatory immune response against P. multocida and the critical involvement of the NLRP3 inflammasome in this activity.

PamulDB: a comprehensive genomic resource for the study of human- and animal-pathogenic Pasteurella multocida.

Pasteurella multocida can infect a wide range of host, including humans and animals of economic importance. Genomics studies on the pathogen have produced a large amount of omics data, which are deposited in GenBank but lacks a dedicated and comprehensive resource for further analysis and integration so that need to be brought together centrally in a coherent and systematic manner. Here we have collected the genomic data for 176 P. multocida strains that are categorized into 11 host groups and 9 serotype groups, and developed the open-access P. multocida Database (PamulDB) to make this resource readily available. The PamulDB implements and integrates Chado for genome data management, Drupal for web content management, and bioinformatics tools like NCBI BLAST, HMMER, PSORTb and OrthoMCL for data analysis. All the P. multocida genomes have been further annotated for search and analysis of homologous sequence, phylogeny, gene ontology, transposon, protein subcellular localization and secreted protein. Transcriptomic data of P. multocida are also selectively adopted for gene expression analysis. The PamulDB has been developing and improving to better aid researchers with identifying and classifying of pathogens, dissecting mechanisms of the pathogen infection and host response.