Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using ¹³C-stable isotope resolved metabolomics and ²H₂O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.     

Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens.     

金黄色葡萄球菌CHIPS基因克隆及原核表达

趋化抑制蛋白(Chemotaxis Inhibitory Protein ofStaphylococcus aureus,CHIPS)是由金黄色葡萄球菌分泌到菌体外的一种蛋白。在感染早期,它能特异性地与中性粒细胞和单核细胞上的C5a受体(C5aR)和fMLP受体(FPR)结合,从而阻止中性粒细胞和单核细胞对C5a和fMLP的结合作用,导致对病原吞噬作用的延迟。人们可以利用CHIPS对C5a-C5aR的阻止作用来研制治疗由C5a诱发的炎症性疾病的药物。将CHIPS作为免疫原防治疾病也将成为新的研究课题。实验成功构建了CHIPS蛋白的原核表达系统,为CHIPS免疫原性研究及蛋白的其他功能研究奠定了基础。     

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.     

Survival of Staphylococcus aureus ST398 in the Human Nose after Artificial Inoculation

There is evidence that MRSA ST398 of animal origin is only capable of temporarily occupying the human nose, and it is therefore, often considered a poor human colonizer.We inoculated 16 healthy human volunteers with a mixture of the human MSSA strain 1036 (ST931, CC8) and the bovine MSSA strain 5062 (ST398, CC398), 7 weeks after a treatment with mupirocin and chlorhexidine-containing soap. Bacterial survival was studied by follow-up cultures over 21 days. The human strain 1036 was eliminated faster (median 14 days; range 2–21 days) than the bovine strain 5062 (median 21 days; range 7–21 days) but this difference was not significant (p = 0.065). The bacterial loads were significantly higher for the bovine strain on day 7 and day 21. 4/14 volunteers (28.6%) showed elimination of both strains within 21 days. Of the 10 remaining volunteers, 5 showed no differences in bacterial counts between both strains, and in the other 5 the ST398 strain far outnumbered the human S. aureus strain. Within the 21 days of follow-up, neither human strain 1036 nor bovine strain 5062 appeared to acquire or lose any mobile genetic elements. In conclusion, S. aureus ST398 strain 5062 is capable of adequately competing for a niche with a human strain and survives in the human nose for at least 21 days.     

Phagocytosis and killing of Staphylococcus aureus by mouse granulocytes after interleukin-3 (IL-3) injection

Interleukin-3 is a multipotential hematopoietic growth factor, which like other colony stimulating factors (CSFs) is effective "in vitro" stimulation of the mature cells function. It was found that IL-3 synergistically with GM-CSF and G-CSF stimulated the proliferation of the granulocytes. Therefore the purpose of this investigation was the evaluation "in vivo" of the influence of IL-3 on the phagocytosis, bactericidal activity, and enzyme activities of granulocytes. IL-3 was injected into mice subcutaneously during 5 days in dose 1 microgram/kg/d. The examination of the percent of cells phagocytizing bacteria (Staphylococcus aureus), NBT test and bactericidal activity, were performed every day and evident increase of the tested parameters was found. Additionally the enzyme activities in primary granules were measured and showed on increase of acid phosphatase and peroxidase activity.     

Characterization of the Starvation-Survival Response of Staphylococcus aureus

The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [³⁵S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.     

Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.     

Effects of Polyethoxyalkyl Phenols on the Leakage of Intracellular Material from Staphylococcus aureus

Polyethoxyoctyl derivatives Triton X45 and Triton X100 induced leakage of cellular materials from Staphylococcus aureus . The total leakage of 260 nm-absorbing material, potassium (K⁺), amino acids and protein depended on the concentrations of surfactant, with K⁺ loss occurring most rapidly. There appeared to be little quantitative correlation between loss of viability and leakage. It is suggested that leakage may reflect changes in membrane permeability resulting in the inability of cells to recover normal functions when placed in a growth-supporting medium.     

Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.     

Induction of stable L-forms of Staphylococcus aureus and Streptococcus faecalis

Stable L-forms were induced from Staphylococcus aureus and Streptococcus faecalis. These formed typical foamy L-colonies and showed large and small round bodies. They grew continuously on routine antibiotic-free nutrient broth and blood agar media for 12 passages without reversion to their parental forms. At different concentrations of penicillin various morphological forms were observed. Effect of sucrose, normal horse serum and penicillin on their adaptation and stabilization is discussed.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.