S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis

Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB

The Burkholderia pseudomallei Type III Secretion System and BopA Are Required for Evasion of LC3-Associated Phagocytosis

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes

Characterisation of Burkholderia pseudomallei type III secretion system III components.

In many intracellular pathogens, the type III secretion system (TTSS) plays an important role in virulence by secreting effector molecules directly across the host cell membrane. These effectors subsequently interact with, and alter, host signalling pathways for the benefit of the pathogen. Burkholderia pseudomallei, an intracellular pathogen that is the causative agent of the potentially fatal disease melioidosis, utilises a TTSS for its survival and replication in both phagocytic and non-phagocytic cells. Although this pathogen contains three TTSS gene clusters, the Type III Secretion System 3 (TTSS3) is critical for bacterial infectivity and pathogenesis. , However, to date, only BopE, BopA and BopC are characterised effectors of the TTSS-3. This research aimed to identify and characterise the putative TTSS3 proteins BapA, BapB and BapC with regard to their possible functions as bacterial effectors involved in either modulation of host cell functions for bacterial survival, replication or escape from host endosomal vacuoles, or the secretion of the other TTSS3 effectors. By using a double cross-over allelic exchange approach, bapA, bapB, bapC and double bapBC mutant strains were generated and assayed for their in vivo and in vitro phenotypes. Competitive growth assays in BALB/c mice showed reduced growth of each of the mutants compared to the wild-type. Furthermore, all showed reduced virulence in the acute mouse infection model, indicating possible roles in bacterial virulence. Complementation was attempted but was unsuccessful. Therefore, independent mutants were constructed. The independent mutants were all tested for virulence in the BALB/c acute model but only the bapA_2 mutant showed reduced virulence compared to the wild-type strain. These data suggest that BapA likely plays a minor role in virulence, although successful complementation is required to conclusively prove this. To determine whether BapA, BapB and BapC were secreted effectors, the TC-FlAsHTM labelling technique was used to monitor the secretion of tetracysteine-tagged fusion proteins. It was demonstrated that BapA and BapC are secreted in vitro. These proteins were secreted in a TTSS3-dependant manner as they were not secreted by mutant B. pseudomallei expressing a non-functional TTSS3. To further investigate any potential involvement of BapA, BapB and BapC in the TTSS3 secretion process, the well-characterised TTSS3 effector BopE was used as a marker to examine TTSS secretion in each of the mutant strains compared to the wild-type and the bopE mutant. The level of transcription of bopE was also assessed in certain strains in order to determine if there was any difference in the transcriptional regulation of this gene. It was demonstrated that, although BapA, BapB and BapC are not required for TTSS function, BapB appears to be necessary for normal secretion of BopE. Therefore, this study defines BapA and BapC as B. pseudomallei TTSS-3 effectors, and BapB as a possible regulator of BopE secretion that may play a role in the pathogenicity of B. pseudomallei

Characterisation of Burkholderia pseudomallei type III secretion system III components.

In many intracellular pathogens, the type III secretion system (TTSS) plays an important role in virulence by secreting effector molecules directly across the host cell membrane. These effectors subsequently interact with, and alter, host signalling pathways for the benefit of the pathogen. Burkholderia pseudomallei, an intracellular pathogen that is the causative agent of the potentially fatal disease melioidosis, utilises a TTSS for its survival and replication in both phagocytic and non-phagocytic cells. Although this pathogen contains three TTSS gene clusters, the Type III Secretion System 3 (TTSS3) is critical for bacterial infectivity and pathogenesis. , However, to date, only BopE, BopA and BopC are characterised effectors of the TTSS-3. This research aimed to identify and characterise the putative TTSS3 proteins BapA, BapB and BapC with regard to their possible functions as bacterial effectors involved in either modulation of host cell functions for bacterial survival, replication or escape from host endosomal vacuoles, or the secretion of the other TTSS3 effectors. By using a double cross-over allelic exchange approach, bapA, bapB, bapC and double bapBC mutant strains were generated and assayed for their in vivo and in vitro phenotypes. Competitive growth assays in BALB/c mice showed reduced growth of each of the mutants compared to the wild-type. Furthermore, all showed reduced virulence in the acute mouse infection model, indicating possible roles in bacterial virulence. Complementation was attempted but was unsuccessful. Therefore, independent mutants were constructed. The independent mutants were all tested for virulence in the BALB/c acute model but only the bapA_2 mutant showed reduced virulence compared to the wild-type strain. These data suggest that BapA likely plays a minor role in virulence, although successful complementation is required to conclusively prove this. To determine whether BapA, BapB and BapC were secreted effectors, the TC-FlAsHTM labelling technique was used to monitor the secretion of tetracysteine-tagged fusion proteins. It was demonstrated that BapA and BapC are secreted in vitro. These proteins were secreted in a TTSS3-dependant manner as they were not secreted by mutant B. pseudomallei expressing a non-functional TTSS3. To further investigate any potential involvement of BapA, BapB and BapC in the TTSS3 secretion process, the well-characterised TTSS3 effector BopE was used as a marker to examine TTSS secretion in each of the mutant strains compared to the wild-type and the bopE mutant. The level of transcription of bopE was also assessed in certain strains in order to determine if there was any difference in the transcriptional regulation of this gene. It was demonstrated that, although BapA, BapB and BapC are not required for TTSS function, BapB appears to be necessary for normal secretion of BopE. Therefore, this study defines BapA and BapC as B. pseudomallei TTSS-3 effectors, and BapB as a possible regulator of BopE secretion that may play a role in the pathogenicity of B. pseudomallei

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness

<i>B. pseudomallei</i>-containing vacuoles are bound by single membranes and the TTSS3 and the effector BopA are required for bacterial escape.

<p>(A–F) Transmission electron micrographs show the intracellular location of <i>B. pseudomallei</i> in RAW 264.7 cells at 2–6 h post infection (p.i.). The scale bar is indicated. Boxed areas are shown as magnified images below each panel. Intracellular bacteria were observed either free in the cytosol and not membrane bound (panel A), or within single-membrane phagosomal compartments (panel B). Only one bacterium was found in a double-membrane compartment (panel C), which could be an autophagosome. Canonical autophagosomes having a double-membrane were observed in infected and uninfected RAW 264.7 cells (panels D–F). Arrows indicate detailed membrane ultrastructure. (G) The percentage of bacteria free in the cytosol of RAW 264.7 cells following infection with <i>B. pseudomallei</i> wild-type, <i>bopA</i> mutant and the <i>bipD</i> mutant at 2, 4, and 6 h p.i. Data represent the mean ± SEM of three separate experiments (n = 100 bacteria). Where shown * indicates p&lt;0.05 relative to the wild-type strain at each time point.</p