Interleukin-10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae.

Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10(-/-) mice). The IL-10(-/-) mice showed increased mortality, higher expression of pro-inflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10(-/-) mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine.

Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans.

BACKGROUND: Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. RESULTS: Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans. CONCLUSION: This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.

A novel sequencing-based vaginal health assay combining self-sampling, HPV detection and genotyping, STI detection, and vaginal microbiome analysis.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman's reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility.

BACKGROUND: Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 1018 times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. In silico prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: Acidithiobacillus ferrooxidans (iron and sulfur oxidizer) A. thiooxidans and A. caldus (sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the Leptospirillum genus that live at pH 1 or below. RESULTS: Acidithiobacilli have predicted FeoB-like Fe(II) and Nramp-like Fe(II)-Mn(II) transporters. They also have 14 different TonB dependent ferri-siderophore transporters of diverse siderophore affinity, although they do not produce classical siderophores. Instead they have predicted novel mechanisms for dicitrate synthesis and possibly also for phosphate-chelation mediated iron uptake. It is hypothesized that the unexpectedly large number and diversity of Fe(III)-uptake systems confers versatility to this group of acidophiles, especially in higher pH environments (pH 4-5) where soluble iron may not be abundant. In contrast, Leptospirilla have only a FtrI-Fet3P-like permease and three TonB dependent ferri-dicitrate siderophore systems. This paucity of iron uptake systems could reflect their obligatory occupation of extremely low pH environments where high concentrations of soluble iron may always be available and were oxidized sulfur species might not compromise iron speciation dynamics. Presence of bacterioferritin in the Acidithiobacilli, polyphosphate accumulation functions and variants of FieF-like diffusion facilitators in both Acidithiobacilli and Leptospirilla, indicate that they may remove or store iron under conditions of variable availability. In addition, the Fe(II)-oxidizing capacity of both A. ferrooxidans and Leptospirilla could itself be a way to evade iron stress imposed by readily available Fe(II) ions at low pH. Fur regulatory sites have been predicted for a number of gene clusters including iron related and non-iron related functions in both the Acidithiobacilli and Leptospirilla, laying the foundation for the future discovery of iron regulated and iron-phosphate coordinated regulatory control circuits. CONCLUSION: In silico analyses of the genomes of acidophilic bacteria are beginning to tease apart the mechanisms that mediate iron uptake and homeostasis in low pH environments. Initial models pinpoint significant differences in abundance and diversity of iron management mechanisms between Leptospirilla and Acidithiobacilli, and begin to reveal how these two groups respond to iron cycling and iron fluctuations in naturally acidic environments and in industrial operations. Niche partitions and ecological successions between acidophilic microorganisms may be partially explained by these observed differences. Models derived from these analyses pave the way for improved hypothesis testing and well directed experimental investigation. In addition, aspects of these models should challenge investigators to evaluate alternative iron management strategies in non-acidophilic model organisms.

Gestational Hypothyroxinemia Imprints a Switch in the Capacity of Astrocytes and Microglial Cells of the Offspring to React in Inflammation.

Hypothyroxinemia (Hpx) is a highly frequent condition characterized by low thyroxine (T4) and normal 3,3',5'-triiodothyronine (T3) and thyroid stimulating hormone (TSH) levels in the blood. Gestational Hpx is closely related to cognitive impairment in the human offspring. In animal models gestational Hpx causes impairment at glutamatergic synapsis, spatial learning, and the susceptibility to suffer strong autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying these phenotypes are unknown. On the other hand, it has been shown that astrocytes and microglia affect the outcome of EAE. In fact, the activation of astrocytes and microglia in the central nervous system (CNS) contributes to EAE progression. Thus, in this work, the reactivity of astrocytes and microglia from rats gestated in Hpx was evaluated aiming to understand whether these cells are targets of gestational Hpx. Interestingly, microglia derived from the offspring gestated in Hpx were less reactive compared to microglia derived from offspring gestated in euthyroidism. Instead, astrocytes derived from the offspring gestated in Hpx were significantly more reactive than the astrocytes from the offspring gestated in euthyroidism. This work contributes with novel information regarding the effects of gestational Hpx over astrocytes and microglia in the offspring. It suggests that astrocyte could react strongly to an inflammatory insult inducing neuronal death in the CNS.

New insights about excisable pathogenicity islands in Salmonella and their contribution to virulence.

Pathogenicity islands (PAIs) are regions of the chromosome of pathogenic bacteria that harbor virulence genes, which were probably acquired by lateral gene transfer. Several PAIs can excise from the bacterial chromosome by site-specific recombination and in this review have been denominated "excisable PAIs". Here, the characteristic of some of the excisable PAIs from Salmonella enterica and the possible role and impact of the excision process on bacterial virulence is discussed. Understanding the role of PAI excision could provide important insights relative to the emergence, evolution and virulence of pathogenic enterobacteria.

Gestational Hypothyroidism Improves the Ability of the Female Offspring to Clear Streptococcus pneumoniae Infection and to Recover From Pneumococcal Pneumonia.

Maternal thyroid hormones are essential for proper fetal development. A deficit of these hormones during gestation has enduring consequences in the central nervous system of the offspring, including detrimental learning and impaired memory. Few studies have shown that thyroid hormone deficiency has a transient effect in the number of T and B cells in the offspring gestated under hypothyroidism; however, there are no studies showing whether maternal hypothyroidism during gestation impacts the response of the offspring to infections. In this study, we have evaluated whether adult mice gestated in hypothyroid mothers have an altered response to pneumococcal pneumonia. We observed that female mice gestated in hypothyroidism have increased survival rate and less bacterial dissemination to blood and brain after an intranasal challenge with Streptococcus pneumoniae. Further, these mice had higher amounts of inflammatory cells in the lungs and reduced production of cytokines characteristic of sepsis in spleen, blood, and brain at 48 hours after infection. Interestingly, mice gestated in hypothyroid mothers had basally increased vascular permeability in the lungs. These observations suggest that gestational hypothyroidism alters the immune response and the physiology of lungs in the offspring, increasing the resistance to respiratory bacterial infections.

Opposing roles of IL-10 in acute bacterial infection.

Interleukin-10 (IL-10) is recognized as an anti-inflammatory cytokine that downmodulates inflammatory immune responses at multiple levels. In innate cells, production of this cytokine is usually triggered after pathogen recognition receptor (PRR) engagement by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patters (DAMPs), as well as by other soluble factors. Importantly, IL-10 is frequently secreted during acute bacterial infections and has been described to play a key role in infection resolution, although its effects can significantly vary depending on the infecting bacterium. While the production of IL-10 might favor host survival in some cases, it may also result harmful for the host in other circumstances, as it can prevent appropriate bacterial clearance. In this review we discuss the role of IL-10 in bacterial clearance and propose that this cytokine is required to recover from infection caused by extracellular or highly pro-inflammatory bacteria. Altogether, we propose that IL-10 drives excessive suppression of the immune response upon infection with intracellular bacteria or in non-inflammatory bacterial infections, which ultimately favors bacterial persistence and dissemination within the host. Thus, the nature of the bacterium causing infection is an important factor that needs to be taken into account when considering new immunotherapies that consist on the modulation of inflammation, such as IL-10. Indeed, induction of this cytokine may significantly improve the host's immune response to certain bacteria when antibiotics are not completely effective.

Conjugal transfer of the pathogenicity island ROD21 in Salmonella enterica serovar Enteritidis depends on environmental conditions.

Unstable pathogenicity islands are chromosomal elements that can be transferred from one bacterium to another. Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic bacterium containing such unstable pathogenicity islands. One of them, denominated ROD21, is 26.5 kb in size and capable of excising from the chromosome in certain culture conditions, as well as during bacterial infection of phagocytic cells. In this study we have evaluated whether ROD21 can be effectively transferred from one bacterium to another. We generated a donor and several recipient strains of S. Enteritidis to carry out transfer assays in liquid LB medium. These assays showed that ROD21 is effectively transferred from donor to recipient strains of S. Enteritidis and S. Typhimurium. When Escherichia coli was used as the recipient strain, ROD21 transfer failed to be observed. Subsequently, we showed that a conjugative process was required for the transfer of the island and that changes in temperature and pH increased the transfer frequency between Salmonella strains. Our data indicate that ROD21 is an unstable pathogenicity island that can be transferred by conjugation in a species-specific manner between Salmonellae. Further, ROD21 transfer frequency increases in response to environmental changes, such as pH and temperature.

Gene elements that regulate Streptococcus pneumoniae virulence and immunity evasion.

Streptococcus pneumoniae is one of the most important aetiological agents of bacterial pneumonia and meningitis in the world. This bacterium can cause severe inflammation of lung tissue and disseminate to the central nervous system. Although B cell activation and antibody secretion is considered one of the most important events in the prevention or clearance of bacterial infection by the host, dendritic cells (DCs) and T cells play a fundamental role in the generation of the protective immunity required to prevent the pathogenesis caused by S. pneumoniae infection. Here we review recent studies that have evaluated the impact of DCs and T cells on S. pneumoniae infection and the gene elements encoding virulence factors used by this bacterium to interfere with the appropriate function of these immune cells. This knowledge could be relevant for generating new prophylactic and therapeutic tools and to prevent the severe infection caused by this pathogen.

Chromosomal excision of a new pathogenicity island modulates Salmonella virulence in vivo.

Although the excision of unstable pathogenicity islands is a phenomenon that has been described for several virulent bacteria, whether this process directly affects the capacity of these microorganisms to cause disease in their hosts remains unknown. Salmonella enterica serovar Enteritidis (S. Enteritidis) is an enterobacterium that harbors several unstable pathogenicity islands that can excise from the main bacterial chromosome. Here we have evaluated whether excision of one of these pathogenicity islands, denominated as Region of Difference 21 (ROD21), is required for S. Enteritidis to cause disease in the host. By means of genetic targeting of the integrase encoded by the ROD21 we have generated S. Enteritidis strains unable to excise ROD21. The failure to excise ROD21 significantly reduced the capacity to cause a lethal disease and to colonize the spleen and liver of mice, as compared to wild type S. Enteritidis. On the contrary, S. Enteritidis strains overexpressing an excisionase protein increased the frequency of ROD21 excision and showed an improved capacity to cause lethal disease in mice. Accordingly, strains unable to excise ROD21 showed an altered expression of genes located in this pathogenicity island. Our results suggest that the genetic excision of the pathogenicity island ROD21 in S. Enteritidis modulates the capacity of this bacterium to cause disease in mice due to a change in the expression of virulence genes.

Excision of an unstable pathogenicity island in Salmonella enterica serovar Enteritidis is induced during infection of phagocytic cells.

The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated "regions of difference" (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells.