The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines

Insights into the Mode of Action of Chitosan as an Antibacterial Compound▿ †

Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death

Rhodococcus equi Virulence-Associated Protein A Is Required for Diversion of Phagosome Biogenesis but Not for Cytotoxicity▿

Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis

Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes

Hansen P, Haubenthal T, Reiter C, et al. Differential Effects of Rhodococcus equi Virulence-Associated Proteins on Macrophages and Artificial Lipid Membranes. Microbiology Spectrum . 2023: e0341722.Virulence-associated protein A (VapA) of Rhodococcus equi is a pathogenicity factor required for the multiplication of virulent R. equi strains within spacious macrophage vacuoles. The production of VapA is characteristic for R. equi isolates from pneumonic foals. VapB and VapN proteins in R. equi isolates from infected pig (VapB) and cattle (VapN) have amino acid sequences very similar to VapA and consequently have been assumed to be its functional correlates. Using model membrane experiments, phagosome pH acidification analysis, lysosome size measurements, protein partitioning, and degradation assays, we provide support for the view that VapA and VapN promote intracellular multiplication of R. equi by neutralizing the pH of the R. equi-containing vacuole. VapB does not neutralize vacuole pH, is not as membrane active as VapA, and does not support intracellular multiplication. This study also shows that the size of the sometimes enormous R. equi-containing vacuoles or the partitioning of purified Vaps into organic phases are not features that have predictive value for virulence of R. equi, whereas the ability of Vaps to increase phagosome pH is coupled to virulence. IMPORTANCE Rhodococcus equi is a major cause of life-threatening pneumonia in foals and occasionally in immunocompromised persons. Virulence-associated protein A (VapA) promotes R. equi multiplication in lung macrophages, which are the major host cells during foal infection. In this study, we compare cellular, biochemical, and biophysical phenotypes associated with VapA to those of VapB (typically produced by isolates from pigs) or VapN (isolates from cattle). Our data support the hypothesis that only some Vaps support multiplication in macrophages by pH neutralization of the phagosomes that R. equi inhabit

<i>In Vivo</i> Identification and Characterization of CD4⁺ Cytotoxic T Cells Induced by Virulent <i>Brucella abortus</i> Infection

<div><p>CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the <i>in vivo</i> identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during <i>Brucella abortus</i> infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against <i>Brucella</i>-infected antigen presenting cells (APC) but not against <i>Mycobacterium</i>-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon <i>Brucella abortus</i> infection. </p> </div

Vacuolar ATPase in Phagosome-Lysosome Fusion

Kissing S, Hermsen C, Repnik U, et al. Vacuolar ATPase in Phagosome-Lysosome Fusion. Journal of Biological Chemistry. 2015;290(22):14166-14180.The vacuolar H+ -ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V-0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V-0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V-0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V-0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes

Bacterial loads and IFN-γ production in <i>Brucella</i> infected mice.

<p>Mice were infected intraperitoneally with 10⁶<i>B. abortus</i> 2308. At 5, 8, 15, 22 and 37 days post-infection, the level of IFN-γ was determined in the sera of mice intraperitoneally infected with <i>Brucella abortus</i> (A). CFUs were determined in the spleens of C57BL/6 infected mice (B). Data are representative of three independent experiments each involving groups of 3 mice.</p

Immunophenotypic characterization of CD4⁺ CTLs upon <i>Brucella</i> infection.

<p>Granzyme B-expressing CD4⁺ T cells from <i>Brucella</i>-infected C57BL/6 mice were analyzed by flow cytometry for the expression of CD25 and the production of IFN-γ at 5 and 8 days post-infection. Numbers in outlined areas indicate percentages of cells. Data are representative of 3 separate experiments each involving groups of 5 mice.</p

Cytolytic capacity of Granzyme B⁺CD4⁺T cells.

<p>Lysis of non-infected and <i>B. abortus</i>-infected macrophages in the presence of CD4⁺ and CD8⁺ T cells isolated from the spleens of BALB/c mice infected for 8 days with <i>Brucella</i> was analysed. Macrophage targets and effector cells were mixed at several effector:target /T ratios and incubated for 4 h. Values given represent relative macrophage lysis as compared to a Tx-100 treated 100% control (A). Parallel samples of heat-killed <i>B. abortus</i>-infected Raw macrophages on coverslips were processed for immunofluorescence microscopy. Representative micrographs show macrophages after 4 h of coculture with CD8⁺ or CD4⁺ effector cells (B). (C) Lysis of non-infected, <i>B. abortus</i>-infected and <i>M. avium</i>-infected macrophages in the presence of CD4⁺ and CD8⁺ T cells isolated from the spleens of BALB/c mice infected for 8 days with <i>Brucella</i> was analysed. Macrophage targets and effector cells were mixed at 1:50 and 1:20 target:effector ratios and incubated for 4 h. Values given represent relative macrophage lysis as compared to a Tx-100 treated 100% control. </p