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

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.

Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE.

BACKGROUND: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. RESULTS: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. CONCLUSION: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.

Laboratory Plasticware Induces Expression of a Bacterial Virulence Factor.

Pollution with microplastic has become a prime environmental concern. The various ways in which human-made polymers and microorganisms interact are little understood, and this is particularly true for microplastic and pathogenic microorganisms. Previous reports demonstrated that expression of central virulence-associated protein A (VapA) of the pathogenic bacterium Rhodococcus equi is shut off at 30°C, whereas it is strongly expressed at 37°C, a temperature which may serve as an intrahost cue. Here, we show that cultivation at 30°C in disposable plastic tubes increases mRNA levels of vapA 70-fold compared to growth in conventional glass tubes. Strong expression of vapA in plastic tubes does not seem to be caused by a compound leaching from plastic but rather by tube surface properties. Expression stimulation during growth in plastic is regulated by the R. equi transcription regulators VirR and VirS, indicating that plastic-induced vapA expression is (co)regulated through the canonical vapA expression pathway. Our observations have important implications for the future analysis and assessment of environmental microplastic contaminations in that they show that, in principle, contact of pathogens with environmental plastic can increase their virulence. IMPORTANCE Millions of tons small plastic pieces (microplastic) find their way into the environment every year. They pose digestive and toxicity problems to various life forms in soil, freshwater, and seawater. Additionally, microplastic offers an opportunity for microorganisms to attach and to become an important part of a "plastisphere community." The significance of our study lies in the documentation of a sharp increase in production of a central virulence factor by a bacterial pathogen when the bacterium is in touch with certain makes of plastic. Although this feature may not reflect an increased health risk in case of this particular soilborne pathogen, our data disclose a new facet of how microplastics can endanger life.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

Gene Expression Profiling of Transcription Factors of Helicobacter pylori under Different Environmental Conditions.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and causes peptic ulcers and gastric carcinoma. H. pylori strain 26695 has a small genome (1.67 Mb), which codes for few known transcriptional regulators that control bacterial metabolism and virulence. We analyzed by qRT-PCR the expression of 16 transcriptional regulators in H. pylori 26695, including the three sigma factors under different environmental conditions. When bacteria were exposed to acidic pH, urea, nickel, or iron, the sigma factors were differentially expressed with a particularly strong induction of fliA. The regulatory genes hrcA, hup, and crdR were highly induced in the presence of urea, nickel, and iron. In terms of biofilm formation fliA, flgR, hp1021, fur, nikR, and crdR were induced in sessile bacteria. Transcriptional expression levels of rpoD, flgR, hspR, hp1043, and cheY were increased in contact with AGS epithelial cells. Kanamycin, chloramphenicol, and tetracycline increased or decreased expression of regulatory genes, showing that these antibiotics affect the transcription of H. pylori. Our data indicate that environmental cues which may be present in the human stomach modulate H. pylori transcription.

COX-2 Inhibition Reduces Brucella Bacterial Burden in Draining Lymph Nodes.

Brucella is a Gram-negative facultative intracellular bacterium responsible for a chronic disease known as brucellosis, the most widespread re-emerging zoonosis worldwide. Establishment of a Th1-mediated immune response characterized by the production of IL-12 and IFNγ is essential to control the disease. Leukotrienes derived from arachidonic acid (AA) metabolism are known to negatively regulate a protective Th1 immune response against bacterial infections. Here, using genomics approaches we demonstrate that Brucella abortus strongly stimulates the prostaglandin (PG) pathway in dendritic cells (DC). We also show an induction of AA production by infected cells. This correlates with the expression of Ptgs2, a gene encoding the downstream cyclooxygenase-2 (COX-2) enzyme in infected DC. By comparing different infection routes (oral, intradermal, intranasal and conjunctival), we identified the intradermal inoculation route as the more potent in inducing Ptgs2 expression but also in inducing a local inflammatory response in the draining cervical lymph nodes (CLN). NS-398, a specific inhibitor of COX-2 enzymatic activity decreased B. melitensis burden in the CLN after intradermal infection. This effect was accompanied by a decrease of Il10 and a concomitant increase of Ifng expression. Altogether, these results suggest that Brucella has evolved to take advantage of the PG pathway in the harsh environment of the CLN in order to persist and subvert immune responses. This work also proposes that novel strategies to control brucellosis may include the use of COX-2 inhibitors.

H-NS Nucleoid Protein Controls Virulence Features of Klebsiella pneumoniae by Regulating the Expression of Type 3 Pili and the Capsule Polysaccharide.

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae.

Vacuolar ATPase in phagosome-lysosome fusion.

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 V0 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 V0 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 V0 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 V0 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.

Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections.

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral routes for means of vaccination.

In vivo identification and characterization of CD4⁺ cytotoxic T cells induced by virulent Brucella abortus infection.

CD4(+) T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4(+) T cells (CD4(+) CTL) during Brucella abortus 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 Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-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 Brucella abortus infection.

BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.

Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for ß-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.

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.

Internal affairs: investigating the Brucella intracellular lifestyle.

Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular 'lifestyle'. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.

Nitric oxide-mediated intracellular growth restriction of pathogenic Rhodococcus equi can be prevented by iron.

Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immunocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosome stage and grows inside a privileged compartment. Here, we show that, in murine macrophages activated with gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h. Whereas infection control of other intracellular pathogens by activated macrophages is executed by enhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control by macrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors, and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated by cotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not in resting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Our results suggest that nitric oxide is central to the restriction of bacterial access to iron in activated macrophages.

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.

Molecular and infection biology of the horse pathogen Rhodococcus equi.

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi-containing vacuoles inside host cells, factors involved in virulence and host resistance and on host-pathogen interaction on organismal and cellular levels.

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.

A mycolyl transferase mutant of Rhodococcus equi lacking capsule integrity is fully virulent.

Rhodococcus equi is a mucoid Gram-positive facultative intracellular pathogen which can cause severe bronchopneumonia in foals and AIDS patients. A polysaccharide capsule which gives R. equi a mucoid appearance has long been suspected to be a virulence factor. Here, we describe a transposome mutant in the gene fbpA of strain R. equi 103 causing absence of a capsular structure. FbpA is a chromosomal gene homologous to antigen 85 (Ag85) mycolyl chain transferase gene of Mycobacterium tuberculosis. The mutant multiplied normally in isolated macrophages, was able to establish the unusual R. equi-containing vacuole in macrophages, was cytotoxic for macrophages, and was virulent in a mouse model. Colonies had a dry appearance on nutrient agar and defective capsule structure. Surprisingly, fbpA mutants cured of the virulence-associated plasmid were found in a phagosome that was more alkaline than that of the corresponding wild-type bacteria, were more cytotoxic and even multiplied to some extent. This study suggests that the capsule is not an important virulence factor of R. equi and that it may even counteract virulence traits.