Opportunistic Intracellular Pathogen Research Articles

Exploring the Chemical Space of Mycobacterial Oxidative Phosphorylation Inhibitors Using Molecular Modeling.

Mycobacteria are opportunistic intracellular pathogens that have plagued humans and other animals throughout history and still are today. They manipulate and hijack phagocytic cells of immune systems, enabling them to occupy this peculiar infection niche. Mycobacteria exploit a plethora of mechanisms to resist antimicrobials (e. g., waxy cell walls, efflux pumps, target modification, biofilms, etc.) thereby evolving into superbugs, such as extensively drug-resistant tuberculosis (XDR TB) bacilli and the emerging pathogenic Mycobacterium abscessus complex. This review summarizes the mechanisms of action of some of the surging antimycobacterial strategies. Exploiting the fact that mycobacteria are obligate aerobes and the differences between their oxidative phosphorylation pathways versus their human counterpart opens a promising avenue for drug discovery. The polymorphism of respiratory complexes across mycobacterial pathogens imposes challenges on the repositioning of antimycobacterial agents to battle the rise in nontuberculous mycobacterial infections. In silico strategies exploiting mycobacterial respiratory machinery data to design novel therapeutic agents are touched upon. The potential druggability of mycobacterial respiratory elements is reviewed. Future research addressing the health challenges associated with mycobacterial pathogens is discussed.

Role of Legionella pneumophila outer membrane vesicles in host-pathogen interaction.

Legionella pneumophila is an opportunistic intracellular pathogen that inhabits artificial water systems and can be transmitted to human hosts by contaminated aerosols. Upon inhalation, it colonizes and grows inside the alveolar macrophages and causes Legionnaires' disease. To effectively control and manage Legionnaires' disease, a deep understanding of the host-pathogen interaction is crucial. Bacterial extracellular vesicles, particularly outer membrane vesicles (OMVs) have emerged as mediators of intercellular communication between bacteria and host cells. These OMVs carry a diverse cargo, including proteins, toxins, virulence factors, and nucleic acids. OMVs play a pivotal role in disease pathogenesis by helping bacteria in colonization, delivering virulence factors into host cells, and modulating host immune responses. This review highlights the role of OMVs in the context of host-pathogen interaction shedding light on the pathogenesis of L. pneumophila. Understanding the functions of OMVs and their cargo provides valuable insights into potential therapeutic targets and interventions for combating Legionnaires' disease.

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases.

Legionella spp. are the causative agents of a severe pneumonia known as Legionnaires' disease. Upon being engulfed by host cells, these environmental bacteria replicate intracellularly in a plasma membrane-derived niche termed Legionella-containing vacuole (LCV) in a way that requires the defective in organelle trafficking/intracellular multiplication (Dot/Icm) protein transporter. Our understanding of interactions between Legionella and its hosts was mostly based on studies of Legionella pneumophila. In this study, we found that the LCVs created by virulent Legionella longbeachae are similarly decorated by polyubiquitinated proteins to those formed by L. pneumophila and that the ubiquitin-proteasome system (UPS) is required for optimal intracellular growth of L. longbeachae. Furthermore, we utilized bioinformatics methods and the ubiquitin-vinylmethyl ester probe to obtain potential deubiquitinases (DUBs) encoded by L. longbeachae. These efforts led to the identification of 9 L. longbeachae DUBs that displayed distinct specificity toward ubiquitin chain types. Among these, LLO_1014 and LLO_2238 are associated with the LCVs and impact the accumulation of polyubiquitinated species on the bacterial phagosome. Moreover, LLO_1014 and LLO_2238 could fully restore the phenotypes associated with Δceg23 (lotB) and Δlem27 (lotC) mutants of L. pneumophila, indicating that these DUBs have similar functions. Together, these results reveal that L. longbeachae uses multiple DUBs to construct an intracellular niche for its replication. IMPORTANCE Legionella spp. are opportunistic intracellular bacterial pathogens that cause Legionnaires' disease. Legionella utilizes the Dot/Icm type IV secretion system to deliver effector protein into host cells to modulate various cellular functions. At least 26 L. pneumophila effectors are known to hijack the host ubiquitin system via diverse mechanisms. L. longbeachae is the second leading cause of Legionnaires' disease worldwide. However, our knowledge about the interactions between L. longbeachae and its hosts is very limited. Here, we found that, similar to L. pneumophila infection, the host ubiquitin proteasome system is also important for the intracellular replication of L. longbeachae. In addition, the bacterial phagosomes harboring L. longbeachae are enriched with polyubiquitinated proteins in a Dot/Icm system-dependent manner. We further identified 9 L. longbeachae proteins that function as DUBs with distinct ubiquitin chain specificity. Of note, several of the phagosome-associated L. longbeachae DUBs regulate the recruitment of polyubiquitinated proteins to the LCV.

Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples.

Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of Legionella.

Ampicillin Treatment of Intracellular Listeria monocytogenes Triggers Formation of Persistent, Drug-Resistant L-Form Cells.

Listeria monocytogenes is an opportunistic intracellular pathogen causing an infection termed listeriosis. Despite the low incidence of listeriosis, the high mortality rate in individuals at risk makes this bacterium one of the most dangerous foodborne pathogens. Reports about a relapse of infection after antibiotic treatment suggest that the bacteria may be able to evade antibiotic treatment and persist as a dormant, antibiotic-tolerant subpopulation. In this study, we observed intracellular generation of antibiotic-resistant L-forms of Listeria monocytogenes following Ampicillin treatment of Listeria monocytogenes infected cells. Detection and identification of intracellular Listeria L-forms was performed by a combination of fluorescence in-situ hybridization and confocal laser scanning microscopy. Using micromanipulation, it was possible to isolate single intracellular L-form cells that following transfer into fresh medium gave rise to pure cultures. In conclusion, the results obtained here provide strong evidence that antibiotic treatment of infected host cells can induce the formation of L-forms from intracellular Listeria monocytogenes. Furthermore, our results suggest that intracellular L-forms persist inside host cells and that they represent viable bacteria, which are still able to grow and proliferate.

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

Tacrolimus exposure windows responsible for Listeria monocytogenes infection susceptibility.

Tacrolimus is widely used to prevent graft rejection after allogeneic transplantation by suppressing T cells in a non-antigen-specific fashion. Global T-cell suppression makes transplant recipients more susceptible to infection, especially infection by opportunistic intracellular pathogens. Infection followed by secondary challenge with the opportunistic intracellular bacterial pathogen, Listeria monocytogenes, was used to probe when tacrolimus most significantly impacts antimicrobial host defense. Tacrolimus-treated mice showed no difference in innate susceptibility following primary infection, whereas susceptibility to secondary challenge was significantly increased. Modifying the timing of tacrolimus initiation with respect to primary infection compared with secondary challenge showed significantly reduced susceptibility in tacrolimus-treated mice where tacrolimus was discontinued prior to secondary challenge. Thus, tacrolimus overrides protection against secondary infection primed by primary infection (and presumably live attenuated vaccines), with the most critical window for tacrolimus-induced infection susceptibility being exposure immediately prior to secondary challenge. These results have important implications for strategies designed to boost antimicrobial T-cell-mediated immunity in transplant recipients.

Mycobacterium avium Modulates the Protective Immune Response in Canine Peripheral Blood Mononuclear Cells.

Mycobacterium avium, an opportunistic intracellular pathogen, is a member of the non-tuberculous mycobacteria species. M. avium causes respiratory disease in immunosuppressed individuals and a wide range of animals, including companion dogs and cats. In particular, the number of infected companion dogs has increased, although the underlying mechanism of M. avium pathogenesis in dogs has not been studied. Therefore, in the present study, the host immune response against M. avium in dogs was investigated by transcriptome analysis of canine peripheral blood mononuclear cells. M. avium was shown to induce different immune responses in canine peripheral blood mononuclear cells at different time points after infection. The expression of Th1-associated genes occurred early during M. avium infection, while that of Th17-associated genes increased after 12 h. In addition, the expression of apoptosis-related genes decreased and the abundance of intracellular M. avium increased in monocyte-derived macrophages after infection for 24 h. These results reveal the M. avium induces Th17 immune response and avoids apoptosis in infected canine cells. As the number of M. avium infection cases increases, the results of the present study will contribute to a better understanding of host immune responses to M. avium infection in companion dogs.

Genetic diversity, virulence and distribution of antimicrobial resistance among Listeria monocytogenes isolated from milk, beef, and bovine farm environment.

Listeria monocytogenes is an opportunistic intracellular foodborne pathogen and is ubiquitous in nature. The occurrence of L. monocytogenes in animal production units coupled with their presence in milk, faeces, feed, water, sewage, and soil is a contributory factor for foodborne listeriosis in humans and animals. The study was aimed to characterize genotype and serogroup of L. monocytogenes recovered from different types of samples and also to study antimicrobial patterns by phenotypic and genotypic methods. Multiplex polymerase chain reaction (PCR) was used for the confirmation of L. monocytogenes, the identification of its serogroup and lineage, and the detection of virulence markers. Enterobacterial repetitive intergenic consensus (ERIC), and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize those isolates, and antimicrobial patterns were studied phenotypically by Kirby-Bauer method and genotypically by PCR. Out of the screened 474 samples (274 milk and 50 each of soil, feed, sewage, and beef), ten L. monocytogenes isolates (milk=8, soil=1, and beef=1) were confirmed by PCR targeting the hlyA gene and found to belong to the 1/2a, 3a serogroup and fall under type II lineage. Virulence potential assessment revealed that all the ten isolates harbored the iap gene while the presence of plcA and plcB genes were noticed in seven and eight isolates respectively. Six isolates from milk were found to group in the same cluster by ERIC and RAPD fingerprinting, suggesting both methods to be efficient molecular typing tools for L. monocytogenes. Genotypic characterization of antimicrobial resistance (AMR) genes revealed that seven isolates were positive for tetM, five for mefA, four for msrA, and one for lnuA genes while none of the isolates showed tetK, ermA, ermB, and lnuB genes. The presence of L. monocytogenes in bovine farm environments coupled with virulence markers, and multidrug resistance from the study area suggest a possible transmission from the environment to humans and animals which needs to be monitored regularly to ensure food safety and the well-being of animals and humans.

Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis

Being opportunistic intracellular pathogens, viruses are dependent on the host for their replication. They hijack host cellular machinery for their replication and survival by targeting crucial cellular physiological pathways, including transcription, translation, immune pathways, and apoptosis. Immediately after translation, the host and viral proteins undergo a process called post-translational modification (PTM). PTMs of proteins involves the attachment of small proteins, carbohydrates/lipids, or chemical groups to the proteins and are crucial for the proteins’ functioning. During viral infection, host proteins utilize PTMs to control the virus replication, using strategies like activating immune response pathways, inhibiting viral protein synthesis, and ultimately eliminating the virus from the host. PTM of viral proteins increases solubility, enhances antigenicity and virulence properties. However, RNA viruses are devoid of enzymes capable of introducing PTMs to their proteins. Hence, they utilize the host PTM machinery to promote their survival. Proteins from viruses belonging to the family: Togaviridae, Flaviviridae, Retroviridae, and Coronaviridae such as chikungunya, dengue, zika, HIV, and coronavirus are a few that are well-known to be modified. This review discusses various host and virus-mediated PTMs that play a role in the outcome during the infection.

Exposure of Mycobacterium avium subsp. homonissuis to Metal Concentrations of the Phagosome Environment Enhances the Selection of Persistent Subpopulation to Antibiotic Treatment.

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic intracellular pathogen causing infections in individuals with chronic lung conditions and patients with immune-deficient disorders. The treatment of MAH infections is prolonged and outcomes many times are suboptimal. The reason for the extended treatment is complex and reflects the inability of current antimicrobials to clear diverse phenotypes of MAH quickly, particularly, the subpopulation of susceptible but drug-tolerant bacilli where the persistent fitness to anti-MAH drugs is stimulated and enhanced by the host environmental stresses. In order to enhance the pathogen killing, we need to understand the fundamentals of persistence mechanism and conditions that can initiate the drug-tolerance phenotype in mycobacteria. MAH can influence the intracellular environment through manipulation of the metal concentrations in the phagosome of infected macrophages. While metals play important role and are crucial for many cellular functions, little is known how vacuole elements influence persistence state of MAH during intracellular growth. In this study, we utilized the in vitro model mimicking the metal concentrations and pH of MAH phagosome at 1 h and 24 h post-infection to distinguish if metals encountered in phagosome could act as a trigger factor for persistence phenotype. Antibiotic treatment of metal mix exposed MAH demonstrates that metals of the phagosome environment can enhance the persistence state, and greater number of tolerant bacteria is recovered from the 24 h metal mix when compared to the viable pathogen number in the 1 h metal mix and 7H9 growth control. In addition, bacterial phenotype induced by the 24 h metal mix increases MAH tolerance to macrophage killing in TNF-α and IFN-γ activated cells, confirming presence of persistent MAH in the 24 h metal mix condition. This work shows that the phagosome environment can promote persistence population in MAH, and that the population differs dependent on a concentration of metals.

Production of IL-17A at Innate Immune Phase Leads to Decreased Th1 Immune Response and Attenuated Host Defense against Infection with Cryptococcus deneoformans.

IL-17A is a proinflammatory cytokine produced by many types of innate immune cells and Th17 cells and is involved in the elimination of extracellularly growing microorganisms, yet the role of this cytokine in the host defense against intracellularly growing microorganisms is not well known. Cryptococcus deneoformans is an opportunistic intracellular growth fungal pathogen that frequently causes fatal meningoencephalitis in patients with impaired immune responses. In the current study, we analyzed the role of IL-17A in the host defense against C. deneoformans infection. IL-17A was quickly produced by γδT cells at an innate immune phase in infected lungs. In IL-17A gene-disrupted mice, clearance of this fungal pathogen and the host immune response mediated by Th1 cells were significantly accelerated in infected lungs compared with wild-type mice. Similarly, killing of this fungus and production of inducible NO synthase and TNF-α were significantly enhanced in IL-17A gene-disrupted mice. In addition, elimination of this fungal pathogen, Th1 response, and expression of IL-12Rβ2 and IFN-γ in NK and NKT cells were significantly suppressed by treatment with rIL-17A. The production of IL-12p40 and TNF-α from bone marrow-derived dendritic cells stimulated with C. deneoformans was significantly suppressed by rIL-17A. In addition, rIL-17A attenuated Th1 cell differentiation in splenocytes from transgenic mice highly expressing TCR for mannoprotein 98, a cryptococcal Ag, upon stimulation with recombinant mannoprotein 98. These data suggest that IL-17A may be involved in the negative regulation of the local host defense against C. deneoformans infection through suppression of the Th1 response.

High Polyphenol Sorghum Extract Attenuates Th2 cytokine Profile and Reduces Growth of Legionella in Raw 264.7 Mouse Macrophage Cell Line

Abstract Objectives 1) To evaluate the changes in inflammatory response induced by sorghum polyphenols in activated macrophages and 2) to evaluate possible efficacy of sorghum polyphenols on an opportunistic intracellular pathogen, Legionella pneumophila (LP). Methods Raw 265.7 cells mouse macrophage cells were treated with sorghum phenolic extract (SPE) under control and activating conditions to evaluate the role of SPE in inflammatory and anti-inflammatory processes. Study measured: nitric oxide production in the supernatant, mRNA using qPCR array of 84 genes in activated macrophages. Morphological changes were observed, and LC3 protein expression was measured to test for autophagy using western blot. NF-kB and STAT3 nuclear translocation was measured using a fractionization kit, followed by western blot. The replication of LP was measured within RAW 264.7 cells and in vitro (in media without cell presence). Cytotoxicity assay and a western blot apoptosis marker caspase-3 were used to evaluate the cytotoxicity of SPE on RAW 264.7 cells. Because LP reproduction within cells is greatly attenuated in the presence of Tumor Necrosis Factor (TNF), LP replication was measured under the presence of TNF neutralizing antibodies. Results SPE decreased nitric oxide production in activated LPS/IFNΥ macrophages although not significantly. SPE attenuated Th2 cytokine response in LPS/IFNΥ activated macrophages by decreasing expression of IL-6 and IL-10 while not changing expression of other inflammatory cytokines. Quantitative PCR data confirmed that genes in the IL-10 and IL-6 pathway were downregulated by cotreatment of SPE and LPS/IFNΥ when compared to LPS/IFNΥ alone. Morphological changes observed exhibited formation of large vacuole like structures. Analysis of LC3 confirmed that autophagy was increased in activated cells treated with SPE. Nuclear fractionization confirmed that STAT3 signaling was attenuated by SPE in activated macrophages. SPE significantly reduced the replication of LP in macrophage cells but not in vitro. The attenuation of LP grown in RAW 264.7 cells was independent of TNF presence. Conclusions This data suggests that sorghum phenolic compounds may have potential pharmaceutical/nutritional uses to combat intracellular pathogens. Funding Sources All funding was provided by the United States Department of Agriculture.

Morin inhibits Listeria monocytogenes virulence in vivo and in vitro by targeting listeriolysin O and inflammation

BackgroundListeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many infections, including meningitis and abortion in humans and animals; thus, L. monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The isolation rate of Listeria monocytogenes in fishery products has always been high. And the pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes. LLO can promote cytosolic bacterial proliferation and help the pathogen evade attacks from the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions.ResultsHere, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization. We also found that morin can effectively alleviate the inflammation induced by Listeria in vivo and in vitro and exerted an obvious protective effect on infected cells and mice.ConclusionsMorin does not possess anti-Listeria activity, neither does it interfere with secretion of LLO. However, morin inhibits oligomerisation of LLO and morin does reduce the inflammation caused during Listeria infection.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Determining the Effects of Legionella pneumophila SidE Effector Proteins on Host Vesicular Trafficking

Legionella pneumophila is an opportunistic intracellular pathogen responsible for Legionnaires’ disease in humans. Upon infection, L. pneumophila forms the Legionella‐containing vacuole (LCV), shielding itself from the host immune response. From the LCV, L. pneumophila uses a specialized secretion system to translocate over 350 effector proteins into the host cell. Effectors target and manipulate host pathways in order to optimize the host’s cellular environment to support L. pneumophila replication (1). One important cellular process targeted during infection is endocytic recycling. Recycling pathways are generally categorized as slow or fast, depending on the time it takes for endocytosed cargo to return to the plasma membrane. Rab11 is a key regulator of the slow endocytic recycling pathway (2) and it moves along microtubules partly through its interaction with Rab11‐FIP1A. It was previously shown that mutation of Rab11‐FIP1A disrupts transferrin recycling (3). In the context of infection by L. pneumophila, our group has shown that transferrin recycling in human macrophages is inhibited early during infection. We hypothesize that the L. pneumophila effectors protein family SidE is at least partly responsible for this defect. Pull‐down assays revealed that interaction between Rab11 and Rab11‐FIP1A is disrupted in the presence of SdeC. In transiently transfected CHO cells, GFP‐Rab11‐FIP1A is found on vesicles in the perinuclear region. However, when GFP‐Rab11‐FIP1A and mCherry‐SdeC are co‐expressed in CHO cells, we observed that GFP‐Rab11‐FIP1A changed its localization to several enlarged structures. We are investigating whether SdeC’s ubiquitination activity is responsible for disrupting interaction of Rab11 with Rab11‐FIP1A. These results will help establish the effects of ubiquitination by SidE effector proteins on the slow recycling pathway.Support or Funding Information

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants.

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious infections and multiple food-borne outbreaks. In this study, we investigated the influence of atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of sigB, rsbR, prfA, gadD, and lmo0799 genes at different treatment time intervals. Further, to ascertain if sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants were similarly affected after 1 min exposure to ACP (p > 0.05), with a difference in response noted only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of treatment (p < 0.05). The results indicate sigB and prfA are important for general stress resistance and biofilm, respectively, loss of these two genes significantly reduced bacterial resistance to ACP treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to cold plasma stress and offers promising insights for optimizing ACP applications.

Optimización de temperatura de anillamiento de PCR multiplex para la detección de Listeria monocytogenes

Listeria monocytogenes es un patógeno intracelular oportunista que se ha convertido en una de las causas más importante de infecciones transmitidas por alimentos en todo el mundo. Debido a la importancia de L. monocytogenes en la salud pública se han descrito muchos protocolos para la identificación y tipificación de este organismo, pero en su mayoría son muy complejos y de larga duración. Por esta razón, se desarrolló un PCR multiplex, para la detección rápida y sensible de esta bacteria en diferentes tipos de muestras. El objetivo del presente estudio fue optimizar la técnica de PCR multiplex para la identificación de L. monocytogenes. Se probaron distintas temperaturas de anillamiento para determinar cuál presenta mayor especificidad, evitando las amplificaciones inespecíficas. Se obtuvo amplificaciones a 57°C y temperaturas menores. Se concluye que la mejor temperatura para tm es la de 57°C.

Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread

ABSTRACT Mycobacterium avium subsp. hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptosis, exit apoptotic cells or via ingestion of infected apoptotic bodies subsequently infect neighboring macrophages. By evaluating 4,000 transposon mutants of M. avium in THP-1 cells, we identified clones that can trigger a new form of early host cell apoptosis, which is only observed upon entry into the “secondary-infected” macrophages. Inactivation of MAVA5_06970 gene lead to significant attenuation in intracellular growth within macrophages and mice, and impaired M. avium to induce rapid apoptosis in the “secondary-infected” cells as measured by Annexin V-FITC detection assay. Complementation of MAVA5_06970 gene corrected the attenuation as well as apoptotic phenotypes. The MAVA5_06970 gene encodes for a secreted protein. Using the pull-down assay and then confirmed with the yeast two-hybrid screen, we found that MAVA5_06970 effector interacts with the Secreted Phosphoprotein 1, the cytokine also known as Osteopontin. This interaction enhances the THP-1 cell apoptosis and, consequently, restricts the production of interleukin-12 that likely may limit the activation of the type I immunity pathway in vivo. This work identified a key virulence effector of M. avium that contributes to the cell-to-cell spread of the pathogen.

Contribution of intercellular adhesion molecule 1 (ICAM-1) to control Mycobacterium avium infection

Mycobacterium avium is a facultative intracellular opportunistic pathogen especially relevant in cases of people living with AIDS. The aim of this study was to evaluate the role of intercellular adhesion molecule 1 (ICAM-1) in the inflammatory response against M. avium infection. Mice deficient for ICAM-1 (ICAM KO) and infected with M. avium presented increased bacterial load in the spleen, liver and lungs compared to C57BL/6. Moreover, ICAM deficient mice presented reduced granuloma area in liver at 30 days post-infection with reduced numbers of lymphocytes and granulocytes. The assessment of in vitro cytokine production by ICAM KO spleen cells showed lower levels of IFN-γ compared to C57BL/6, whereas TNF-α remained unaltered. Additionally, the production of IFN-γ in liver and spleen tissues was also diminished in ICAM-1 KO mice. Interestingly, a persistent reduction in IFN-γ production was observed in CD3+NK1.1+ cells of ICAM-1 deficient mice compared to wild-type animals. Together, these results demonstrate the importance of ICAM-1 in the efficient control of M. avium infection and granuloma formation and highlights its role on CD3+NK1.1+ cell population as important for IFN-γ production during infection.