Keeping the host alive - lessons from obligate intracellular bacterial pathogens.

Mammals have evolved sophisticated host cell death signaling pathways as an important immune mechanism to recognize and eliminate cell intruders before they establish their replicative niche. However, intracellular bacterial pathogens that have co-evolved with their host have developed a multitude of tactics to counteract this defense strategy to facilitate their survival and replication. This requires manipulation of pro-death and pro-survival host signaling pathways during infection. Obligate intracellular bacterial pathogens are organisms that absolutely require an eukaryotic host to survive and replicate, and therefore they have developed virulence factors to prevent diverse forms of host cell death and conserve their replicative niche. This review encapsulates our current understanding of these host-pathogen interactions by exploring the most relevant findings of Anaplasma spp., Chlamydia spp., Rickettsia spp. and Coxiella burnetii modulating host cell death pathways. A detailed comprehension of the molecular mechanisms through which these obligate intracellular pathogens manipulate regulated host cell death will not only increase the current understanding of these difficult-to-study pathogens but also provide insights into new tools to study regulated cell death and the development of new therapeutic approaches to control infection.

Impact of STING Inflammatory Signaling during Intracellular Bacterial Infections.

The early detection of bacterial pathogens through immune sensors is an essential step in innate immunity. STING (Stimulator of Interferon Genes) has emerged as a key mediator of inflammation in the setting of infection by connecting pathogen cytosolic recognition with immune responses. STING detects bacteria by directly recognizing cyclic dinucleotides or indirectly by bacterial genomic DNA sensing through the cyclic GMP-AMP synthase (cGAS). Upon activation, STING triggers a plethora of powerful signaling pathways, including the production of type I interferons and proinflammatory cytokines. STING activation has also been associated with the induction of endoplasmic reticulum (ER) stress and the associated inflammatory responses. Recent reports indicate that STING-dependent pathways participate in the metabolic reprogramming of macrophages and contribute to the establishment and maintenance of a robust inflammatory profile. The induction of this inflammatory state is typically antimicrobial and related to pathogen clearance. However, depending on the infection, STING-mediated immune responses can be detrimental to the host, facilitating bacterial survival, indicating an intricate balance between immune signaling and inflammation during bacterial infections. In this paper, we review recent insights regarding the role of STING in inducing an inflammatory profile upon intracellular bacterial entry in host cells and discuss the impact of STING signaling on the outcome of infection. Unraveling the STING-mediated inflammatory responses can enable a better understanding of the pathogenesis of certain bacterial diseases and reveal the potential of new antimicrobial therapy.

Autophagy and Its Interaction With Intracellular Bacterial Pathogens.

Cellular responses to stress can be defined by the overwhelming number of changes that cells go through upon contact with and stressful conditions such as infection and modifications in nutritional status. One of the main cellular responses to stress is autophagy. Much progress has been made in the understanding of the mechanisms involved in the induction of autophagy during infection by intracellular bacteria. This review aims to discuss recent findings on the role of autophagy as a cellular response to intracellular bacterial pathogens such as, Streptococcus pyogenes, Mycobacterium tuberculosis, Shigella flexneri, Salmonella typhimurium, Listeria monocytogenes, and Legionella pneumophila, how the autophagic machinery senses these bacteria directly or indirectly (through the detection of bacteria-induced nutritional stress), and how some of these bacterial pathogens manage to escape from autophagy.

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism.

The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.

Education to a Healthy Lifestyle Improves Symptoms and Cardiovascular Risk Factors – AsuRiesgo Study / Educação para um Estilo de Vida Saudável Melhora Sintomas e Fatores de Risco Cardiovasculares – Estudo AsuRiesgo

Background: Cardiovascular diseases are the current leading causes of death and disability globally. Objective: To assess the effects of a basic educational program for cardiovascular prevention in an unselected outpatient population. Methods: All participants received an educational program to change to a healthy lifestyle. Assessments were conducted at study enrollment and during follow-up. Symptoms, habits, ATP III parameters for metabolic syndrome, and American Heart Association’s 2020 parameters of cardiovascular health were assessed. Results: A total of 15,073 participants aged ≥ 18 years entered the study. Data analysis was conducted in 3,009 patients who completed a second assessment. An improvement in weight (from 76.6 ± 15.3 to 76.4 ± 15.3 kg, p = 0.002), dyspnea on exertion NYHA grade II (from 23.4% to 21.0%) and grade III (from 15.8% to 14.0%) and a decrease in the proportion of current active smokers (from 3.6% to 2.9%, p = 0.002) could be documented. The proportion of patients with levels of triglycerides > 150 mg/dL (from 46.3% to 42.4%, p < 0.001) and LDL cholesterol > 100 mg/dL (from 69.3% to 65.5%, p < 0.001) improved. A ≥ 20% improvement of AHA 2020 metrics at the level graded as poor was found for smoking (-21.1%), diet (-29.8%), and cholesterol level (-23.6%). A large dropout as a surrogate indicator for low patient adherence was documented throughout the first 5 visits, 80% between the first and second assessments, 55.6% between the second and third assessments, 43.6% between the third and fourth assessments, and 38% between the fourth and fifth assessments. Conclusion: A simple, basic educational program may improve symptoms and modifiable cardiovascular risk factors, but shows low patient adherence. .

Fundamentos: As doenças cardiovasculares são, atualmente, as maiores causas de óbito e incapacitação em todo o mundo. Objetivos: Avaliar os efeitos de um programa educativo básico para prevenção cardiovascular em uma população de pacientes ambulatoriais não selecionados. Métodos: Todos os participantes frequentaram um programa educativo de mudança para um estilo de vida saudável. Foram realizadas avaliações à admissão no estudo e durante o acompanhamento. Foram avaliados sintomas, hábitos, parâmetros do ATP III para síndrome metabólica e parâmetros da American Heart Association 2020 para saúde cardiovascular. Resultados: Foram incluídos no estudo 15.073 participantes com idade ≥ 18 anos. Foi feita a análise de dados dos 3.009 pacientes que completaram a segunda avaliação. Foram documentados perda de peso (de 76,6 ± 15,3 para 76,4 ± 15,3 kg, p = 0,002), melhora da dispneia aos esforços graus II-NYHA (de 23,4% para 21,0%) e III (de 15,8% para 14,0%), e redução na proporção de fumantes ativos atuais (de 3,6% para 2,9%, p = 0,002). Houve melhora na proporção de pacientes com níveis de triglicérides > 150 mg/dL (de 46,3% para 42,4%, p < 0,001) e de colesterol LDL > 100 mg/dL (de 69,3% para 65,5%, p < 0,001). Houve melhora ≥ 20% na métrica AHA 2020 no nível classificado como ruim para tabagismo (-21,1%), alimentação (-29,8%), e nível de colesterol (23,6%). Foi documentada grande evasão como indicador substituto para baixa adesão de paciente nas primeiras 5 consultas, sendo 80% entre a primeira e a segunda avaliação, 55,6% entre a segunda e a terceira, 43,6% entre a terceira e a quarta, e 38% entre a quarta e a quinta. Conclusão: Um programa educativo básico e simples pode melhorar os sintomas e fatores de risco cardiovasculares modificáveis, mas conta com pouca adesão por parte dos pacientes. .

Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi.

BACKGROUND: Naturally occurring coinfections of pathogens have been reported in salmonids, but their consequences on disease resistance are unclear. We hypothesized that 1) coinfection of Caligus rogercresseyi reduces the resistance of Atlantic salmon to Piscirickettsia salmonis; and 2) coinfection resistance is a heritable trait that does not correlate with resistance to a single infection. METHODOLOGY: In total, 1,634 pedigreed Atlantic salmon were exposed to a single infection (SI) of P. salmonis (primary pathogen) or coinfection with C. rogercresseyi (secondary pathogen). Low and high level of coinfection were evaluated (LC = 44 copepodites per fish; HC = 88 copepodites per fish). Survival and quantitative genetic analyses were performed to determine the resistance to the single infection and coinfections. MAIN FINDINGS: C. rogercresseyi significantly increased the mortality in fish infected with P. salmonis (SI mortality = 251/545; LC mortality = 544/544 and HC mortality = 545/545). Heritability estimates for resistance to P. salmonis were similar and of medium magnitude in all treatments (h2SI = 0.23 ± 0.07; h2LC = 0.17 ± 0.08; h2HC = 0.24 ± 0.07). A large and significant genetic correlation with regard to resistance was observed between coinfection treatments (rg LC-HC = 0.99 ± 0.01) but not between the single and coinfection treatments (rg SI-LC = -0.14 ± 0.33; rg SI-HC = 0.32 ± 0.34). CONCLUSIONS/SIGNIFICANCE: C. rogercresseyi, as a secondary pathogen, reduces the resistance of Atlantic salmon to the pathogen P. salmonis. Resistance to coinfection of Piscirickettsia salmonis and Caligus rogercresseyi in Atlantic salmon is a heritable trait. The absence of a genetic correlation between resistance to a single infection and resistance to coinfection indicates that different genes control these processes. Coinfection of different pathogens and resistance to coinfection needs to be considered in future research on salmon farming, selective breeding and conservation.

Pre-exposure of Mycobacterium tuberculosis-infected macrophages to crystalline silica impairs control of bacterial growth by deregulating the balance between apoptosis and necrosis.

Inhalation of crystalline silica (CS) particles increases the risk of pulmonary tuberculosis; however, the precise mechanism through which CS exposure facilitates Mycobacterium tuberculosis (Mtb) infection is unclear. We speculate that macrophage exposure to CS deregulates the cell death pathways that could explain, at least in part, the association observed between exposure to CS and pulmonary tuberculosis. We therefore established an in vitro model in which macrophages were exposed to CS and then infected with Mtb. Expression of surface markers was analyzed by flow cytometry, JNK1/2, ASK1, caspase 9, P-p38, Bcl-2 and Mcl-1 were analyzed by Western blot, and cytokines by ELISA. Our results show that exposure to CS limits macrophage ability to control Mtb growth. Moreover, this exposure reduced the expression of TLR2, Bcl-2 and Mcl-1, but increased that of JNK1 and ASK1 molecules in the macrophages. Finally, when the pre-exposed macrophages were infected with Mtb, the concentrations of TNFα, IL-1ß and caspase-9 expression increased. This pro-inflammatory profile of the macrophage unbalanced the apoptosis/necrosis pathway. Taken together, these data suggest that macrophages exposed to CS are sensitized to cell death by MAPK kinase-dependent signaling pathway. Secretion of TNF-α and IL-1ß by Mtb-infected macrophages promotes necrosis, and this deregulation of cell death pathways may favor the release of viable bacilli, thus leading to the progression of tuberculosis.

Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

TGF-ß-mediated sustained ERK1/2 activity promotes the inhibition of intracellular growth of Mycobacterium avium in epithelioid cells surrogates.

Transforming growth factor beta (TGF-ß) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-ß production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-ß and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-ß produced by ECs are sufficient to induce a self-sustaining autocrine TGF-ß signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-ß to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-ß receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-ß produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-ß receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-ß production. In summary, our findings indicate that the amplitude of TGF-ß signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize.

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants' magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplasts and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.

Effect of intracellular Wolbachia on interspecific crosses between Drosophila melanogaster and Drosophila simulans.

Wolbachia are bacteria that live inside the cells of a large number of invertebrate hosts and are transmitted from infected females to their offspring. Their presence is associated with cytoplasmic incompatibility in several species of Drosophila. Cytoplasmic incompatibility results when the sperm of infected males fertilize eggs of uninfected females, causing more or less intense embryonic mortality (unidirectional incompatibility). This phenomenon also appears in crosses between populations infected with different Wolbachia strains (bidirectional incompatibility). The influence of Wolbachia infection on host populations has attracted attention as a potentially rapid mechanism for development of reproductive isolation and subsequent speciation. We examined the influence of this bacterium on reproductive isolation in interspecific crosses between Drosophila melanogaster and D. simulans. We found that Wolbachia infection negatively affected these two species in homospecific crosses. However, in interspecific crosses, it only influenced sexual isolation, as infected females more frequently hybridized than females free of infection; postzygotic reduction of fitness (bidirectional cytoplasmic incompatibility) was not detected. This would be explained by the existence of several modes of rescue systems in these two species, reducing cytoplasmic incompatibility between them. Wolbachia does not appear to cause reproductive isolation between these two species.

Productive infection of Piscirickettsia salmonis in macrophages and monocyte-like cells from rainbow trout, a possible survival strategy.

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS), an endemic disease which causes significant losses in salmon production. This intracellular bacterium is normally cultured in salmonid epithelial cell lines inducing characteristic cytopathic effects (CPEs). In this study we demonstrate that P. salmonis is able to infect, survive, replicate, and propagate in the macrophages/monocytes cell line RTS11 derived from rainbow trout spleen, without inducing the characteristic CPEs and the host cells showing the same expression levels as non-infected control cell. On the other hand, bacteria were capable of expressing specific proteins within infected cells. Infected macrophages cease proliferation and a fraction of them detached from the plate, transform to non-adhesive, monocyte-like cells with proliferative activity. Productive infection of P. salmonis into salmonid macrophage/monocyte cells in culture provides an excellent model for the study of host-pathogen interactions, almost unknown in the case of P. salmonis. Our results suggest that the infection of cells from the salmonid innate immune system without inducing an important cell death response should lead to the persistence of the bacteria and consequently their dissemination to other tissues, favoring the evasion of the first line of defense against pathogens.

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplast s and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.(AU)

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants' magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplast s and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.

Autophagy in intracellular bacterial infection.

Numerous pathogens have developed the capacity to invade host cells to be protected from components of the systemic immune system. However, once in the host cells they utilize sophisticated strategies to avoid the powerful machinery built by the cells to kill invading pathogens. In the last few years cumulative evidence indicates that autophagy is one of the most remarkable tools of the intracellular host cell defense machinery that bacteria must confront upon cell invasion. However, several pathogens subvert the autophagic pathway and, manipulate this process at the molecular level, as a strategy to establish a persistent infection. In this review we have summarized the interaction between autophagy and different bacterial pathogens including those that take advantage of the host cell autophagy, allowing successful colonization, as well as those microorganisms which are controlled by autophagy as part of the innate surveillance mechanism.