Rafael Prados-Rosales

Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi.

Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus, Mycobacterium tuberculosis and Cryptococcus neoformans. These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis. In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB

Nitrogen limitation has been proposed to act as a signal for infectious development in plant pathogens. Here, the authors show that the preferred nitrogen source ammonium represses a set of virulence-related functions in the vascular wilt fungus Fusarium oxysporum. They also identify two key elements of the nitrogen response pathway: the conserved protein kinase TOR and the bZIP factor MeaB. During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source–independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the ΔmeaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.

Ab binding alters gene expression in Cryptococcus neoformans and directly modulates fungal metabolism

Abs facilitate humoral immunity via the classical mechanisms of opsonization, complement activation, Ab-dependent cellular cytotoxicity, and toxin/viral neutralization. There is also evidence that some Abs mediate direct antimicrobial effects. For example, Ab binding to the polysaccharide capsule of the human pathogenic fungus Cryptococcus neoformans promotes opsonization but also inhibits polysaccharide release and biofilm formation. To investigate whether Ab binding affects C. neoformans directly, we analyzed fungal gene expression after binding of protective and nonprotective mAbs. The 2 IgM Abs and 1 IgG1 Ab tested each induced different changes in gene expression. The protective IgG1 mAb upregulated genes encoding proteins involved in fatty acid synthesis, the protective IgM mAb downregulated genes encoding proteins required for protein translation, and the nonprotective IgM mAb had modest effects on gene expression. Differences in gene expression correlated with mAb binding to different locations of the capsule. Of the 3 Abs tested, the protective IgG1 mAb bound to C. neoformans closest to the cell wall, produced specific differences in the pattern of phosphorylated proteins, caused changes in lipid metabolism, and resulted in increased susceptibility to the antifungal drug amphotericin B. These results suggest what we believe to be a new mode of action for Ab-mediated immunity and raise the possibility that immunoglobulins mediate cross talk between microbes and hosts through their effects on microbial metabolism.

Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae

UNLABELLED Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications. BIOLOGICAL SIGNIFICANCE Pneumococcus is one of the leading causes of bacterial pneumonia worldwide in children and the elderly, being responsible for high morbidity and mortality rates in developing countries. The augment of pneumococcal disease in developed countries has raised major public health concern, since the difficulties to treat these infections due to increasing antibiotic resistance. Vaccination is still the best way to combat pneumococcal infections. One of the mechanisms that bacterial pathogens use to combat the defense responses of invaded hosts is the production and release of extracellular vesicles derived from the outer surface. Little is known about this phenomenon in Gram-positives. We show that pneumococcus produces membrane-derived vesicles particularly enriched in lipoproteins. We also show the utility of pneumococcal vesicles as a new type of vaccine, as they induce protection in immunized mice against infection with a virulent strain. This work will contribute to understand the role of these structures in important biological processes such as host-pathogen interactions and prevention of human disease.

Release of mycobacterial antigens.

Mycobacterium tuberculosis has evolved from a Mycobacterium canettii‐like progenitor pool into one of the most successful and widespread human pathogens. The pathogenicity of M. tuberculosis is linked to its ability to secrete/export/release selected mycobacterial proteins, and it is also established that active release of mycobacterial antigens is a prerequisite for strong immune recognition. Recent research has enabled mycobacterial secretion systems and vesicle‐based release of mycobacterial antigens to be elucidated, which together with host‐related specificities constitute key variables that determine the outcome of infection. Here, we discuss recently discovered, novel aspects on the nature and the regulation of antigen release of the tuberculosis agent with particular emphasis on the biological characterization of mycobacteria‐specific ESX/type VII secretion systems and their secreted proteins, belonging to the Esx, PE, and PPE categories. The importance of specific mycobacterial antigen release is probably best exemplified by the striking differences observed between the cellular events during infection with the ESX‐1‐deficient, attenuated Mycobacterium bovis BCG compared to the virulent M. tuberculosis, which are clearly important for design of more specific diagnostics and more efficient vaccines.

Mycobacterial Membrane Vesicles Administered Systemically in Mice Induce a Protective Immune Response to Surface Compartments of Mycobacterium tuberculosis

ABSTRACT Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation. This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.

A PR-1-like Protein of Fusarium oxysporum Functions in Virulence on Mammalian Hosts

Background: Pathogenesis-related (PR-1-like) proteins are widely conserved in eukaryotes, but their biological function is unknown. Results: Knockout or site-directed mutagenesis of fpr1 encoding a secreted PR-1-like protein in the fungal pathogen Fusarium oxysporum impairs virulence on mice. Conclusion: Secreted PR-1-like proteins are important for fungal infection of mammals. Significance: We show the first genetic evidence for a biological function of the predicted active site of PR-1-like proteins. The pathogenesis-related PR-1-like protein family comprises secreted proteins from the animal, plant, and fungal kingdoms whose biological function remains poorly understood. Here we have characterized a PR-1-like protein, Fpr1, from Fusarium oxysporum, an ubiquitous fungal pathogen that causes vascular wilt disease on a wide range of plant species and can produce life-threatening infections in immunocompromised humans. Fpr1 is secreted and proteolytically processed by the fungus. The fpr1 gene is required for virulence in a disseminated immunodepressed mouse model, and its function depends on the integrity of the proposed active site of PR-1-like proteins. Fpr1 belongs to a gene family that has expanded in plant pathogenic Sordariomycetes. These results suggest that secreted PR-1-like proteins play important roles in fungal pathogenicity.

Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

Significance Bacteria stimulate host cells in part via secreted products, some of which are packaged in membrane vesicles (MV). MV released by the major human pathogen Mycobacterium tuberculosis (Mtb) carry lipoprotein LpqH, a major agonist for host Toll-like receptor 2 (TLR2). This study identifies a gene, rv0431, which appears to regulate mycobacterial MV formation, and therefore we suggest it be named “vesiculogenesis and immune response regulator” (virR). This gene encodes a protein that includes a unique fold, as determined by NMR spectroscopy, and a disordered domain suggestive of participation in a higher-order complex. By restraining the release of most of the material released by Mtb that activates host cells through TLR2, VirR reduces Mtb’s immunostimulatory potential and increases its virulence. Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host–pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named “vesiculogenesis and immune response regulator.”