RESUMO
Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Bacteriemia , COVID-19 , Pneumonia Bacteriana , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Animais , Carbapenêmicos/farmacologia , Humanos , Imunidade Inata , Ferro/metabolismo , Lipocalina-2/genética , Lipocalina-2/metabolismo , Camundongos , Pandemias , Sideróforos/metabolismoRESUMO
Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial toward S. aureus, but rather, enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produce higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Antibacterianos , Cálcio/metabolismo , Canais de Cálcio , Proteínas de Ligação ao Cálcio , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Neutrófilos/metabolismo , Staphylococcus aureus/metabolismo , SuperóxidosRESUMO
The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Metionina Sulfóxido Redutases/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/imunologia , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Peróxido de Hidrogênio/metabolismo , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/imunologia , Camundongos , Viabilidade Microbiana/imunologia , Mutação , Oxirredução , Estresse Oxidativo , Staphylococcus aureus/genéticaRESUMO
Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9-/- and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9-/- mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9-/- mice accumulated significantly more IL-13+IL-5+CD4+ T-helper type 2 cells. S100A9-/- mice also accumulated a significantly lower proportion of CD4+ T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4+ T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4+ Treg cell function.
Assuntos
Alveolite Alérgica Extrínseca/imunologia , Calgranulina B/fisiologia , Complexo Antígeno L1 Leucocitário/fisiologia , Pulmão/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Imunidade Adaptativa , Alérgenos/toxicidade , Alternaria/imunologia , Alveolite Alérgica Extrínseca/etiologia , Alveolite Alérgica Extrínseca/patologia , Animais , Hiper-Reatividade Brônquica/etiologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar/química , Calgranulina A/biossíntese , Calgranulina A/genética , Calgranulina B/genética , Citocinas/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Imunoglobulina E/imunologia , Inflamação , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Eosinofilia Pulmonar/etiologia , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologia , Organismos Livres de Patógenos EspecíficosRESUMO
Landscape disturbances can alter habitat structure and resource availability, often inducing physiological responses by organisms to cope with the changing conditions. Quantifying the endocrine stress response through measurement of glucocorticoids has become an increasingly common method for determining how organisms physiologically respond to challenges imposed by their environment. We tested the hypothesis that Eastern Fence Lizards cope with fire disturbance effects by modulating their secretion of corticosterone (CORT). We measured the baseline and stress-induced plasma CORT of male Eastern Fence Lizards in a chronosequence of fire-altered habitats (recently burned, recovering from burn, and unburned). Although habitat use by lizards differed among burn treatments, including differences in use of canopy cover, leaf litter, and vegetation composition, we did not detect a significant effect of fire-induced habitat alteration on plasma CORT concentration or on body condition. In addition, we found no effect of blood draw treatment (baseline or stress-induced), body temperature, body condition, or time taken to collect blood samples on concentration of plasma CORT. Low intensity burns, which are typical of prescribed fire, may not be a sufficient stressor to alter CORT secretion in Eastern Fence Lizards (at least during the breeding season). Instead, lizards may avoid allostatic overload using behavioral responses and by selecting microsites within their environment that permit thermoregulatory opportunities necessary for optimal performance and energy assimilation.
RESUMO
Zinc (Zn) is an essential trace metal required for all forms of life, but is toxic at high concentrations. While the toxic effects of high levels of Zn are well documented, the mechanism of cell death appears to vary based on the study and concentration of Zn. Zn has been proposed as an anti-cancer treatment against non-small cell lung cancer (NSCLC). The goal of this analysis was to determine the effects of Zn on metabolism and cell death in A549 cells. Here, high throughput multi-omics analysis identified the molecular effects of Zn intoxication on the proteome, metabolome, and transcriptome of A549 human NSCLC cells after 5 min to 24 h of Zn exposure. Multi-omics analysis combined with additional experimental evidence suggests Zn intoxication induces ferroptosis, an iron and lipid peroxidation-dependent programmed cell death, demonstrating the utility of multi-omics analysis to identify cellular response to intoxicants.
Assuntos
Ferroptose/efeitos dos fármacos , Pulmão/patologia , Zinco/toxicidade , Células A549 , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Genômica , Humanos , NAD/biossíntese , Necrose , Ligação Proteica/efeitos dos fármacos , Fatores de TempoRESUMO
Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.
Assuntos
Infecções por Mycoplasma/veterinária , Tartarugas/microbiologia , Animais , Anticorpos Antibacterianos/análise , California , NevadaRESUMO
A seminatural, factorial-design experiment was used to quantify dynamics of the pathogen Mycoplasma agassizii and upper respiratory tract disease in the Mojave desert tortoise (Gopherus agassizii) over 2 years. Groups of initially healthy animals were separated into serologically positive (seropositive), seronegative, and artificially infected groups and paired into 23 pens. We found no evidence of long-term immune protection to M. agassizii or of immunological memory. Initially seronegative, healthy tortoises experienced an equal amount of disease when paired with other seronegative groups as when paired with seropositive and artificially infected groups-suggesting that recrudescence is as significant as transmission in introducing disease in individuals in this host-pathogen system. Artificially infected groups of tortoises showed reduced levels of morbidity when paired with initially seronegative animals-suggesting either a dilution effect or a strong effect of pathogen load in this system. Physiological dynamics within the host appear to be instrumental in producing morbidity, recrudescence, and infectiousness, and thus of population-level dynamics. We suggest new avenues for studying diseases in long-lived ectothermic vertebrates and a shift in modeling such diseases.