Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Bordetella parapertussis adenylate cyclase toxin promotes the bacterial survival to the encounter with macrophages.

B. parapertussis is a whooping cough etiological agent, whose incidence in the population has increased remarkably. Virulence factors involved in the bacterial infection, however, remain poorly investigated. We here studied the role of adenylate cyclase (CyaA), the main toxin of B. parapertussis, in the outcome of the bacterial interaction with macrophages. Our results showed that B. parapertussis CyaA intoxicates human macrophages, prevents bacterial phagocytosis and precludes phago-lysosomal fusion eventually promoting the bacterial survival to the encounter with these immune cells. Accordingly, we found that B. parapertussis CyaA induces the transcriptional downregulation of host genes encoding for antimicrobial peptides, proteins involved in bacterial intracellular killing, and the pro-inflammatory cytokine TNF-α, while induces the upregulation of the anti-inflammatory cytokine IL-10. Together with previous reports suggesting a protective role of B. parapertussis CyaA against neutrophils bactericidal activity, the results of this study suggest a central role of CyaA in B. parapertussis immune evasion and persistence.

Intracellular replication of Inquilinus limosus in bronchial epithelial cells.

Inquilinus limosus is an emerging multi-resistant opportunistic pathogen documented mainly in cystic fibrosis patients. Infection with I. limosus is accompanied by either an acute respiratory exacerbation or a progressive loss of pulmonary function. This study examined the interaction of Inquilinus limosus with the bronquial human epithelial cell line 16HBE14o-. Almost 100% of the bacteria that attached to the bronquial cells were found internalized and located in acidic LAMP2 positive compartments. According to confocal studies combined with antibiotic protection assays, I. limosus is able to survive and eventually replicate in these compartments. I. limosus was found nontoxic to cells and did not induce neither IL-6 nor IL-8 cytokine production, a characteristic that may help the bacteria to evade host immune response. Overall, this study indicates that I. limosus displays pathogenic properties based on its ability to survive intracellularly in epithelial cells eventually leading to antibiotic failure and chronic infection.

Bordetella pertussis outer membrane vesicles as virulence factor vehicles that influence bacterial interaction with macrophages.

Gram-negative pathogenic bacteria constitutively shed outer membrane vesicles (OMVs) which play a significant role in the host-pathogen interaction, eventually determining the outcome of the infection. We previously found that Bordetella pertussis, the etiological agent of whooping cough, survives the innate interaction with human macrophages remaining alive inside these immune cells. Adenylate cyclase (CyaA), one of the main toxins of this pathogen, was found involved in the modulation of the macrophage defense response, eventually promoting bacterial survival within the cells. We here investigated whether B. pertussis OMVs, loaded with most of the bacterial toxins and CyaA among them, modulate the macrophage response to the bacterial infection. We observed that the pre-incubation of macrophages with OMVs led to a decreased macrophage defense response to the encounter with the bacteria, in a CyaA dependent way. Our results suggest that CyaA delivered by B. pertussis OMVs dampens macrophages protective function by decreasing phagocytosis and the bactericidal capability of these host cells. By increasing the chances of bacterial survival to the innate encounter with the macrophages, B. pertussis OMVs might play a relevant role in the course of infection, promoting bacterial persistence within the host and eventually, shaping the whole infection process.

The intracellular phase of extracellular respiratory tract bacterial pathogens and its role on pathogen-host interactions during infection.

PURPOSE OF REVIEW: An initial intracellular phase of usually extracellular bacterial pathogens displays an important strategy to hide from the host's immune system and antibiotics therapy. It helps the bacteria, including bacterial pathogens of airway diseases, to persist and eventually switch to a typical extracellular infection. Several infectious diseases of the lung are life-threatening and their control is impeded by intracellular persistence of pathogens. Thus, molecular adaptations of the pathogens to this niche but also the host's response and potential targets to interfere are of relevance. Here we discuss examples of historically considered extracellular pathogens of the respiratory airway where the intracellular survival and proliferation is well documented, including infections by Staphylococcus aureus, Bordetella pertussis, Haemophilus influenzae, Pseudomonas aeruginosa, and others. RECENT FINDINGS: Current studies focus on bacterial factors contributing to adhesion, iron acquisition, and intracellular survival as well as ways to target them for combatting the bacterial infections. SUMMARY: The investigation of common and specific mechanisms of pathogenesis and persistence of these bacteria in the host may contribute to future investigations and identifications of relevant factors and/or bacterial mechanisms to be blocked to treat or improve prevention strategies.

Hfq modulates global protein pattern and stress response in Bordetella pertussis.

B. pertussis is the etiological agent of whooping cough, a highly contagious respiratory disease which remains uncontrolled worldwide. Understanding how this pathogen responds to the environmental changes and adapts to different niches found inside the host might contribute to gain insight into bacterial pathogenesis. Comparative analyses of previous transcriptomic and proteomic data suggested that post-transcriptional regulatory mechanisms modulate B. pertussis virulence in response to iron availability. Iron scarcity represents one of the major stresses faced by bacterial pathogens inside the host. In this study, we used gel-free nanoLC-MS/MS-based proteomics to investigate whether Hfq, a highly conserved post-transcriptional regulatory protein, is involved in B. pertussis adaptation to low iron environment. To this end, we compared the protein profiles of wild type B. pertussis and its isogenic hfq deletion mutant strain under iron-replete and iron-depleted conditions. Almost of 33% of the proteins identified under iron starvation was found to be Hfq-dependent. Among them, proteins involved in oxidative stress tolerance and virulence factors that play a key role in the early steps of host colonization and bacterial persistence inside the host cells. Altogether these results suggest that Hfq shapes the infective phenotype of B. pertussis. SIGNIFICANCE: In the last years, it became evident that post-transcriptional regulation of gene expression in ba cteria plays a central role in host-pathogen interactions. Hfq is a bacterial protein that regulates gene expression at post-transcriptional level found pivotal in the establishment of successful infections. In this study, we investigated the role of Hfq in Bordetella pertussis response to iron starvation, one of the main stresses imposed by the host. The data demonstrate that Hfq regulates the abundance of a significant number of B. pertussis proteins in response to iron starvation. Among them, virulence factors and proteins involved in oxidative stress tolerance, key players in host colonization and intracellular bacterial survival. Altogether, our results suggest a relevant role of Hfq in B. pertussis adaptation to the different niches found inside the host eventually granting bacterial pathogenesis.

Shotgun proteomic analysis of Bordetella parapertussis provides insights into the physiological response to iron starvation and potential new virulence determinants absent in Bordetella pertussis.

Bordetella parapertussis is one of the pathogens that cause whooping cough. Even though its incidence has been rising in the last decades, this species remained poorly investigated. This study reports the first extensive proteome analysis of this bacterium. In an attempt to gain some insight into the infective phenotype, we evaluated the response of B. parapertussis to iron starvation, a critical stress the bacteria face during infection. Among other relevant findings, we observed that the adaptation to this condition involves significant changes in the abundance of two important virulence factors of this pathogen, namely, adenylate cyclase and the O-antigen. We further used the proteomic data to search for B. parapertussis proteins that are absent or classified as pseudogenes in the genome of Bordetella pertussis to unravel differences between both whooping cough causative agents. Among them, we identified proteins involved in stress resistance and virulence determinants that might help to explain the differences in the pathogenesis of these species and the lack of cross-protection of current acellular vaccines. Altogether, these results contribute to a better understanding of B. parapertussis biology and pathogenesis. SIGNIFICANCE: Whooping cough is a reemerging disease caused by both Bordetella pertussis and Bordetella parapertussis. Current vaccines fail to induce protection against B parapertussis and the incidence of this species has been rising over the years. The proteomic analysis of this study provided relevant insights into potential virulence determinants of this poorly-studied pathogen. It further identified proteins produced by B. parapertussis not present in B. pertussis, which might help to explain both the differences on their respective infectious process and the current vaccine failure. Altogether, the results of this study contribute to the better understanding of B. parapertussis pathogenesis and the eventual design of improved preventive strategies against whooping cough.

A Bordetella pertussis MgtC homolog plays a role in the intracellular survival.

Bordetella pertussis, the causative agent of whooping cough, has the capability to survive inside the host cells. This process requires efficient adaptation of the pathogen to the intracellular environment and the associated stress. Among the proteins produced by the intracellular B. pertussis we identified a protein (BP0414) that shares homology with MgtC, a protein which was previously shown to be involved in the intracellular survival of other pathogens. To explore if BP0414 plays a role in B. pertussis intracellular survival a mutant strain defective in the production of this protein was constructed. Using standard in vitro growth conditions we found that BP0414 is required for B. pertussis growth under low magnesium availability or low pH, two environmental conditions that this pathogen might face within the host cell. Intracellular survival studies showed that MgtC is indeed involved in B. pertussis viability inside the macrophages. The use of bafilomycin A1, which inhibits phagosome acidification, abolished the survival defect of the mgtC deficient mutant strain suggesting that in intracellular B. pertussis the role of MgtC protein is mainly related to the bacterial adaptation to the acidic conditions found inside the of phagosomes. Overall, this work provides an insight into the importance of MgtC in B. pertussis pathogenesis and its contribution to bacterial survival within immune cells.

Novel Guanidine Compound against Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species.

Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF). Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to treat and highlight an urgent need for the development of new class of agents to combat these infections. In this work, a small library comprising thiourea and guanidine derivatives with low molecular weight was designed; these derivatives were studied as antimicrobial agents against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and 2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide and meropenem. The role that different substituents exert in the antimicrobial activity has been determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI) > 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that H-BDF can be considered as a promising antimicrobial agent.

Bordetella pertussis modulates human macrophage defense gene expression.

Bordetella pertussis, the etiological agent of whooping cough, still causes outbreaks. We recently found evidence that B. pertussis can survive and even replicate inside human macrophages, indicating that this host cell might serve as a niche for persistence. In this work, we examined the interaction of B. pertussis with a human monocyte cell line (THP-1) that differentiates into macrophages in culture in order to investigate the host cell response to the infection and the mechanisms that promote that intracellular survival. To that end, we investigated the expression profile of a selected number of genes involved in cellular bactericidal activity and the inflammatory response during the early and late phases of infection. The bactericidal and inflammatory response of infected macrophages was progressively downregulated, while the number of THP-1 cells heavily loaded with live bacteria increased over time postinfection. Two of the main toxins of B. pertussis, pertussis toxin (Ptx) and adenylate cyclase (CyaA), were found to be involved in manipulating the host cell response. Therefore, failure to express either toxin proved detrimental to the development of intracellular infections by those bacteria. Taken together, these results support the relevance of host defense gene manipulation to the outcome of the interaction between B. pertussis and macrophages.

Proteome analysis of Bordetella pertussis isolated from human macrophages.

Previous studies have shown that B. pertussis survives inside human macrophages in non-acidic compartments with characteristics of early endosomes. In order to gain new insight into the biology of B. pertussis survival in host cells, we have analyzed the adaptation of the bacterial proteome during intracellular infection. The proteome of B. pertussis 3 h and 48 h after infection of human macrophage-like THP-1 cells was examined by nano-liquid chromatography combined with tandem MS and compared to the protein profile of extracellular B. pertussis growing in the same cell culture medium. Compared with extracellular bacteria, almost 300 proteins out of 762 identified proteins displayed altered levels in intracellular B. pertussis. Functional analyses of the proteins displaying altered abundance revealed enrichment of proteins involved in stress response, iron uptake, cellular metabolism, transcriptional regulation, and virulence. To our knowledge, this is the first analysis of the B. pertussis proteome during adaptation to the intramacrophage environment and the data provide new clues for understanding B. pertussis adaptation and pathogenesis. BIOLOGICAL SIGNIFICANCE: B. pertussis is a respiratory pathogen that has adapted exclusively to the human host. Despite high vaccination rates, whooping cough remains a serious threat to human health and its incidence has been increasing in recent years in vaccinated populations. The mechanisms that allow this pathogen to evade immune clearance, persist in the host, and cause a prolonged paroxysmal cough are still poorly understood. Recent studies regarding B. pertussis survival inside host cells and the cellular response to this bacterial infection indicate that B. pertussis may have an intracellular phase during infection which probably contributes to persistence and vaccine failure. In this study we provide the first global proteome profile of B. pertussis within macrophages. The data provide novel insights into the adaptive responses elicited by these bacteria for physiological adaptation to the host environment.

Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism, virulence, and defense response.

One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel-free nanoLC-MS/MS-based comparative proteome analysis of Bordetella pertussis grown under iron-excess and iron-depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty-seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly-hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two-component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host-pathogen interaction.

Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

Bordetella pertussis iron regulated proteins as potential vaccine components.

Bordetella pertussis is the etiologic agent of whooping cough, an illness whose incidence has been increasing over the last decades. Pertussis reemergence despite high vaccination coverage, together with the recent isolation of circulating strains deficient in some of the vaccine antigens, highlight the need for new vaccines. Proteins induced under physiological conditions, such as those required for nutrient acquisition during infection, might represent good targets for better preventive strategies. By mean of serological proteome analysis we identified two novel antigens of B. pertussis potentially involved in iron acquisition during host colonization. We had previously demonstrated that one of them, designated IRP1-3, is protective against pertussis infection in mice. In the present study, we show that the other antigen, named AfuA (BP1605), is a highly antigenic protein, exposed on the bacterial surface, conserved among clinical isolates and expressed during infection. Immunization of mice with the recombinant AfuA induced opsonophagocytic antibodies which could explain the protection against B. pertussis infection conferred by mice immunization with rAfuA. Importantly, we found that the addition of rAfuA and rIRP1-3 proteins to the commercial three pertussis components acellular vaccine significantly increased its protective activity. Taken together, our results point at these two antigens as potential components of a new generation of acellular vaccines.

Bordetella parapertussis survives the innate interaction with human neutrophils by impairing bactericidal trafficking inside the cell through a lipid raft-dependent mechanism mediated by the lipopolysaccharide O antigen.

Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen.

Identification of a new protective antigen of Bordetella pertussis.

Antigenic proteins whose expression is induced under iron starvation, an environmental condition that bacterial pathogens have to face during colonization, might be potential candidates for improved vaccine. By mean of immune proteomics we identified novel antigens of Bordetella pertussis maximally expressed under iron limitation. Among them, Bp1152 (named as IRP1-3) showed a particularly strong reaction with human IgG purified from pooled sera of pertussis-infected individuals. Computer analysis showed IRP1-3 as a dimeric membrane protein potentially involved in iron uptake. Experimental data revealed the surface-exposure of this protein and showed its increase under iron starvation to be independent of bacterial virulence phase. Immunization of mice with the recombinant IRP1-3 resulted in a strong antibody response. These antibodies not only recognized the native protein on bacterial surface but also promote effective bacterial phagocytosis by human PMN, a key protecting activity against this pathogen. Accordingly, IRP1-3 proved protective against B. pertussis infection in mouse model. Expression of IRP1-3 was found conserved among clinical isolates of B. pertussis and positively regulated by iron starvation in these strains. Taken together these results suggest that this protein might be an interesting novel vaccine candidate.

Intracellular trafficking of Bordetella pertussis in human macrophages.

Although Bordetella pertussis has been observed to survive inside macrophages, its ability to resist or evade degradation in phagolysosomes has not been defined. We here investigated the trafficking of B. pertussis upon entry into human macrophages. During the first hours following phagocytosis, a high percentage of bacteria were destroyed within acidic compartments positive for the lysosome-associated membrane proteins (LAMP). However, roughly one-fourth of the bacteria taken up evade this initial killing event, remaining in nonacidic compartments. Forty-eight hours after infection, the number of intracellular bacteria per cell increased, suggesting that B. pertussis is capable of replicating in this type of compartment. Viable bacteria accumulated within phagosomal compartments positive for the early endosomal marker Rab5 but not the late endosomal marker LAMP. Moreover, B. pertussis-containing phagosomes acquired exogenously added transferrin, indicating that intracellular bacteria have access to extracellular components and essential nutrients via the host cell recycling pathway. Overall, these results suggest that B. pertussis survives and eventually replicates in compartments with characteristics of early endosomes, potentially contributing to its extraordinary ability to persist within hosts and populations.

Cholesterol-dependent attachment of human respiratory cells by Bordetella pertussis.

Bordetella pertussis is a re-emerging human respiratory pathogen whose infectious process is not fully understood, hampering the design of effective vaccines. The nature of bacterial attachment to host cells is a key event in the outcome of the infection. However, host cell receptors involved in B. pertussis colonization of the respiratory tract are still under investigation. Here, we report that cholesterol-rich domains are involved in B. pertussis adhesion to epithelial cells. Treatment of A549 cells with cholesterol-sequestering drugs such as methyl-beta-cyclodextrin, nystatin, or filipin resulted in a significant decrease of B. pertussis attachment. Confocal laser microscopy studies showed B. pertussis associated with cholesterol-rich domains. Accordingly, B. pertussis was found in detergent-resistant membrane domain fractions isolated from bacterial-infected A549 cells. Our results indicate a main role of filamentous hemagglutinin, an environmentally regulated virulence factor, in this interaction, and a specific affinity for cholesterol, one of the major components of tracheal secretions, which might additionally contribute to the effective colonization of the respiratory tract.

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils.

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-beta-cyclodextrin (MbetaCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.

Iron stress increases Bordetella pertussis mucin-binding capacity and attachment to respiratory epithelial cells.

Whooping cough is a reemerging infectious disease of the respiratory tract caused by Bordetella pertussis. The incomplete understanding of the molecular mechanisms of host colonization hampers the efforts to control this disease. Among the environmental factors that commonly determine the bacterial phenotype, the concentration of essential nutrients is of particular importance. Iron, a crucial and scarce nutrient in the natural environment of B. pertussis, has been found to induce substantial phenotypic changes in this pathogen. However, the relevance of this phenotype for the interaction with host cells was never investigated. Using an in vitro model for bacterial attachment, it was shown that the attachment capacity of B. pertussis to epithelial respiratory cells is enhanced under iron stress conditions. Attachment is mediated by iron-induced surface-exposed proteins with sialic acid-binding capacity. The results further suggest that some of these iron-induced surface-associated proteins are immunogenic and may represent attractive vaccine candidates.