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Bordetella pertussis causes the acute disease whooping cough and survives only in the human respiratory tract, while Bordetella bronchiseptica causes long-term, chronic infections in a broad range of mammals and can also survive in extra-host environments. These bacteria produce a nearly identical set of virulence factors, including adenylate cyclase toxin (ACT), a protein that is modified by the addition of acyl chains. Acylation is required for ACT to cause hemolysis and for efficient intoxication of host cells in vitro . We found that ACT acylation is also important, but not absolutely required, during infection. We also discovered differences in ACT production, acylation, and secretion between B. bronchiseptica and B. pertussis that may contribute to the different virulence strategies of these species. This study highlights the advantage of conducting comparative analyses between closely related species to better understand the evolution of virulence.

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of . Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing strain completely protected mice against lethal challenge with wild-type virulent , indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Bordetella pertussis , Bordetella parapertussis and Bordetella bronchiseptica are closely related Gram-negative β-proteobacteria that colonize the respiratory tracts of mammals. B. pertussis is a strict human pathogen of recent evolutionary origin and is the primary etiologic agent of whooping cough. B. parapertussis can also cause whooping cough, and B. bronchiseptica causes chronic respiratory infections in a wide range of animals. We sequenced the genomes of B. bronchiseptica RB50 (5,338,400 bp; 5,007 predicted genes), B. parapertussis 12822 (4,773,551 bp; 4,404 genes) and B. pertussis Tohama I (4,086,186 bp; 3,816 genes). Our analysis indicates that B. parapertussis and B. pertussis are independent derivatives of B. bronchiseptica -like ancestors. During the evolution of these two host-restricted species there was large-scale gene loss and inactivation; host adaptation seems to be a consequence of loss, not gain, of function, and differences in virulence may be related to loss of regulatory or control functions.

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

Bordetella bronchiseptica is a potential zoonotic pathogen, which mainly causes respiratory diseases in humans and a variety of animal species. B. bronchiseptica is one of the important pathogens isolated from rabbits in Fujian Province. However, the knowledge of the epidemiology and characteristics of the B. bronchiseptica in rabbits in Fujian Province is largely unknown. In this study, 219 B. bronchiseptica isolates recovered from lung samples of dead rabbits with respiratory diseases in Fujian Province were characterised by multi-locus sequencing typing, screening virulence genes and testing antimicrobial susceptibility. The results showed that the 219 isolates were typed into 11 sequence types (STs) including five known STs (ST6, ST10, ST12, ST14 and ST33) and six new STs (ST88, ST89, ST90, ST91, ST92 and ST93) and the ST33 (30.14%, 66/219), ST14 (26.94%, 59/219) and ST12 (16.44%, 36/219) were the three most prevalent STs. Surprisingly, all the 219 isolates carried the five virulence genes (fhaB, prn, cyaA, dnt and bteA) in the polymerase chain reaction screening. Moreover, the isolates were resistant to cefixime, ceftizoxime, cefatriaxone and ampicillin at rates of 33.33%, 31.05%, 11.87% and 3.20%, respectively. This study showed the genetic diversity of B. bronchiseptica in rabbits in Fujian Province, and the colonisation of the human-associated ST12 strain in rabbits in Fujian Province. The results might be useful for monitoring the epidemic strains, developing preventive methods and preventing the transmission of epidemic strains from rabbits to humans.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B . pertussis , the causal agent of human whooping cough, and B . bronchiseptica , which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B . bronchiseptica , which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B . pertussis , which evolved from a B . bronchiseptica -like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1 , ctaCDFGE1 , and cyoABCD1 . To test the hypothesis that the three cytochrome oxidases encoded within the B . pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B . bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo . No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B . pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B . bronchiseptica producing only the three B . pertussis -conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.

Bordetella bronchiseptica and Bordetella pertussis are two closely related respiratory pathogens that employ their T3SS injectisome to deliver the BteA effector into host cells. In this study, we visualized the needle tip filament of their T3SS injectisome, a structure formed by the Bsp22 protein. We demonstrate that during Bordetella cultivation in Stainer-Scholte medium, Bsp22 filaments are abundant and can dynamically extend up to several micrometers in length through the incorporation of new subunits at their distal ends. In contrast, these filaments become shorter and/or less abundant during infection of host cells. This reduction correlates with decreased bsp22 mRNA expression and lower Bsp22 protein levels, while the levels of bscD mRNA, which encodes the inner membrane ring protein of the injectisome, remain stable. These results highlight the adaptability of the Bordetella T3SS injectisome and show how its tip filament structure changes in response to different environments.

Introduction Cunninghamella elegans infections cause rare and severe mucormycosis. Bordetella bronchiseptica and Pneumocystis jirovecii relate to pneumonia. They are all clinically uncommon pathogens and no reports of co-infections have been reported. Case presentation Here we present a case of a 67-year-old male patient who initially presented with fever, chills, and mild cough. B. bronchiseptica , P. jirovecii , Aspergillus fumigatus , and human alphaherpesvirus 1 (HSV1) were detected by clinical metagenomic next-generation sequencing (mNGS) of his bronchoalveolar lavage fluid (BLAF). Despite receiving anti-infective treatment, the patient rapidly developed respiratory failure and was transferred to the intensive care unit. Subsequent mNGS testing further revealed the presence of C. elegans , indicating that different pathogens played dominant roles at various stages of the disease progression. The routine culture also identified several of the above pathogens, but the results were reported much later than those of mNGS. Eventually, imaging findings and symptoms of the patient improved with comprehensive antibiotic coverage, and he was transferred to a lower-level hospital for rehabilitation treatment. Conclusions This is the first detailed report of the combined infection of B. bronchiseptica , P. jirovecii , and C. elegans . During the treatment process, we also observed rare and unusual neurological side effects: visual and auditory hallucinations, restlessness, and aphasia. Also, the case indicates that traditional methods are insufficient for the etiological diagnosis needs of critical and severe patient populations, and timely use of mNGS should be recommended.