Bordetella pertussis outer membrane vesicles impair neutrophil bactericidal activity.

Neutrophils constitute the primary defense against bacterial infections, yet certain pathogens express virulence factors that enable them to subvert neutrophils-mediated killing. Outer membrane vesicles (OMVs) have emerged as a secretory system through which bacteria deliver virulence factors to host cells. OMVs from Bordetella pertussis, the etiological agent of whooping cough, are loaded with most of bacterial virulence factors, including CyaA, which plays a key role in B. pertussis evasion of neutrophils bactericidal activity. In our study, we investigated the role of B. pertussis OMVs in bacterial interaction with neutrophils. We observed that interaction of OMVs with neutrophils led to a decrease in the expression of cell surface CR3 and FcγRs, an effect dependent on the CyaA toxin delivered by these vesicles. This decreased receptor expression led to reduced bacterial uptake by neutrophils, irrespective of the presence of opsonic antibodies. Moreover, CyaA delivered by OMVs hindered intracellular bactericidal trafficking, promoting bacterial intracellular survival. When both bacteria and OMVs were opsonized, competition between opsonized OMVs and B. pertussis for FcγRs on neutrophils led to a significant decrease in bacterial uptake. Overall, our findings suggest that B. pertussis OMVs promote bacterial survival to the encounter with neutrophils in both naïve and immunized individuals.

BPP0974 is a Bordetella parapertussis adhesin expressed in the avirulent phase, implicated in biofilm formation and intracellular survival.

B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis.

Bordetella pertussis targets the basolateral membrane of polarized respiratory epithelial cells, gets internalized, and survives in intracellular locations.

The airway epithelial barrier is a continuous highly organized cell layer that separates the exterior from the underlying mucosal tissue, preventing pathogen invasion. Several respiratory pathogens have evolved mechanisms to compromise this barrier, invade and even reside alive within the epithelium. Bordetella pertussis is a persistent pathogen that infects the human airway epithelium, causing whooping cough. Previous studies have shown that B. pertussis survives inside phagocytic and nonphagocytic cells, suggesting that there might be an intracellular stage involved in the bacterial infectious process and/or in the pathogen persistence inside the host. In this study we found evidence that B. pertussis is able to survive inside respiratory epithelial cells. According to our results, this pathogen preferentially attaches near or on top of the tight junctions in polarized human bronchial epithelial cells and disrupts these structures in an adenylate cyclase-dependent manner, exposing their basolateral membrane. We further found that the bacterial internalization is significantly higher in cells exposing this membrane compared with cells only exposing the apical membrane. Once internalized, B. pertussis mainly remains in nondegradative phagosomes with access to nutrients. Taken together, these results point at the respiratory epithelial cells as a potential niche of persistence.

Adenylate cyclase toxin of Bordetella parapertussis disrupts the epithelial barrier granting the bacterial access to the intracellular space of epithelial cells.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.

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.

Human macrophage polarization shapes B. pertussis intracellular persistence.

We previously demonstrated that Bordetella pertussis, the etiologic agent of whooping cough, is able to survive inside human macrophages. The aim of this study was to examine the influence of macrophage polarization in the development of B. pertussis intracellular infections. To this end, primary human monocytes were differentiated into M1, M2a, or M2c macrophages and further infected with B. pertussis. Infected M1 macrophages showed a proinflammatory response evidenced by the production of TNF-α, IL-12p70, and IL-6. Conversely, infection of M2a and M2c macrophages did not induce TNF-α, IL-12p70, nor IL-6 at any time postinfection but showed a significant increase of M2 markers, such as CD206, CD163, and CD209. Interestingly, anti-inflammatory cytokines, like IL-10 and TGF-ß, were induced after infection in the 3 macrophage phenotypes. B. pertussis phagocytosis by M1 macrophages was lower than by M2 phenotypes, which may be ascribed to differences in the expression level of B. pertussis docking molecules on the surface of the different phenotypes. Intracellular bactericidal activity was found to be significantly higher in M1 than in M2a or M2c cells, but live bacteria were still detected within the 3 phenotypes at the late time points after infection. In summary, this study shows that intracellular B. pertussis is able to survive regardless of the macrophage activation program, but its intracellular survival proved higher in M2 compared with the M1 macrophages, being M2c the best candidate to develop into a niche of persistence for B. pertussis.

Fibrillar conformation of an apolipoprotein A-I variant involved in amyloidosis and atherosclerosis.

BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.

Clinical Approach to Flash Glucose Monitoring: An Expert Recommendation.

The flash glucose monitoring (FGM) system FreeStyle Libre® is a device that measures interstitial glucose in a very simple way and indicates direction and speed of glucose change. This allows persons with diabetes to prevent hypoglycemic and hyperglycemic events. Scientific evidence indicates that the system can improve glycemic control and quality of life. To obtain the maximum benefit, it is necessary to properly handle glucose values and trends. Due to the generalization of the system use, the purpose of the document is to provide recommendations for the optimal use of the device, not only in the management of glucose values and trends but also in the prevention of hypoglycemia, actuation in exercise, special situations, and retrospective analysis of the glucose data, among others.

Junin Virus Triggers Macrophage Activation and Modulates Polarization According to Viral Strain Pathogenicity.

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies.

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.

Bordetella parapertussis Circumvents Neutrophil Extracellular Bactericidal Mechanisms.

B. parapertussis is a whooping cough etiological agent with the ability to evade the immune response induced by pertussis vaccines. We previously demonstrated that in the absence of opsonic antibodies B. parapertussis hampers phagocytosis by neutrophils and macrophages and, when phagocytosed, blocks intracellular killing by interfering with phagolysosomal fusion. But neutrophils can kill and/or immobilize extracellular bacteria through non-phagocytic mechanisms such as degranulation and neutrophil extracellular traps (NETs). In this study we demonstrated that B. parapertussis also has the ability to circumvent these two neutrophil extracellular bactericidal activities. The lack of neutrophil degranulation was found dependent on the O antigen that targets the bacteria to cell lipid rafts, eventually avoiding the fusion of nascent phagosomes with specific and azurophilic granules. IgG opsonization overcame this inhibition of neutrophil degranulation. We further observed that B. parapertussis did not induce NETs release in resting neutrophils and inhibited NETs formation in response to phorbol myristate acetate (PMA) stimulation by a mechanism dependent on adenylate cyclase toxin (CyaA)-mediated inhibition of reactive oxygen species (ROS) generation. Thus, B. parapertussis modulates neutrophil bactericidal activity through two different mechanisms, one related to the lack of proper NETs-inducer stimuli and the other one related to an active inhibitory mechanism. Together with previous results these data suggest that B. parapertussis has the ability to subvert the main neutrophil bactericidal functions, inhibiting efficient clearance in non-immune hosts.

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.

Bordetella parapertussis survives inside human macrophages in lipid raft-enriched phagosomes.

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.

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 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.

Differential effects of HNF-1α mutations associated with familial young-onset diabetes on target gene regulation.

Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype.

Cannabinoid CB1 receptors are expressed by parietal cells of the human gastric mucosa.

Experimental data suggest that the endogenous cannabinoid system is involved in gastric function in different animal species. In most of them, CB(1) receptors have been localized on vagal terminals innervating the external wall of the stomach. We aimed at studying the putative presence and distribution of these receptors in the human gastric mucosa. To this end, we first performed Western blotting, RT-PCR, in situ hybridization, and immunohistochemical analysis of CB(1) protein distribution in biopsy samples of healthy individuals. To determine the precise cell populations expressing CB(1) receptors, we performed double immunofluorescence plus confocal microscopy analysis of the same samples. Our results show that CB(1) receptors are present in the gastric epithelium of the mucosa. Specifically, they are expressed by a subpopulation of mucosal cells, the acid-secreting parietal cells, as shown by double immunohistochemical staining and by their differential abundance in subregions of the gastric mucosa. These results reinforce the notion of a prominent role for the endocannabinoid system in the gastric function in humans and postulate the use of cannabinoid CB(1) receptors in parietal cells as new therapeutic targets for the regulation of gastric acid production.

A Spanish founder mutation in the chloride channel gene, CLCNKB, as a cause of atypical Bartter syndrome in adult age.

BACKGROUND: Mutations in the chloride channel gene, CLCNKB, usually cause classic Bartter syndrome (cBS) or a mixed Bartter-Gitelman phenotype in the first years of life. METHODS: We report an adult woman with atypical BS caused by a homozygous missense mutation, A204T, in the CLCNKB gene, which has previously been described as the apparently unique cause of cBS in Spain. RESULTS: The evaluation of this patient revealed an overlap of phenotypic features ranging from severe biochemical and systemic disturbances typical of cBS to scarce symptoms and diagnosis in the adult age typical of Gitelman syndrome. The tubular disease caused a dramatic effect on mental, growth and puberal development leading to low IQ, final short stature and abnormal ovarian function. Furthermore, low serum PTH concentrations with concomitant nephrocalcinosis and normocalcaemia were observed. Both ovarian function and serum PTH levels were normalized after treatment with cyclooxygenase inhibitors. CONCLUSIONS: The present report confirms a weak genotype-phenotype correlation in patients with CLCNKB mutations and supports the founder effect of the A204T mutation in Spain. In our country, the genetic diagnosis of adult patients with hereditary hypokalaemic tubulopathies should include a screening of A204T mutation in the CLCNKB gene.