Comparative physiological study of the wild type and the small colony variant of Pseudomonas aeruginosa 20265 under controlled growth conditions

Small-colony variants (SCVs) of Pseudomonas aeruginosa are often found in chronically infected airways of patients suffering from cystic fibrosis. These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections. Nevertheless, the behavior of SCVs under varied availability of O₂ and iron, two key variables relevant to the lung environment of CF patients and pathogenicity of P. aeruginosa, has not been systematically studied so far. In this work, the effects of O₂ and iron were comparatively studied for a CF P. aeruginosa wild type (WT) strain and its SCV phenotype in a real-time controlled cultivation system. Significant differences in the behavior of these strains were observed and quantified. In general, SCV exhibited a higher fitness than the WT toward aerobic conditions. Under iron rich condition, and despite less release of total extracellular proteins, absence of flagellin and lower siderophore production, the SCV cells grown at fully aerobic conditions showed a higher specific growth rate and a significantly higher cytotoxicity in comparison with the WT cells. The strains behaved also differently towards iron limitation. The phenomena of limited O₂ transfer from the gas to the liquid phase and enhancement of formation of virulence factors under conditions of iron limitation were much more profound in the SCV culture than in the WT culture. These results have important implications for better understanding the pathogenicity of P. aeruginosa and its small-colony variants.     

Characteristics of Staphylococcus aureus, isolated from airways of cystic fibrosis patients, and their small colony variants

The colonization of respiratory tract by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF), especially in pediatric patients. The formation of small colony variants (SCVs), which produce reduced amounts of alpha-toxin, is one of the proposed ways of staphylococcal accommodation in an intracellular niche. The aim of the present study was to compare some properties of S. aureus SCVs and their parent strains. A site-directed S. aureus hemB mutant and parent strain 8325-4 were included in the study (control pair). Normal and SCV strain pairs from CF patients as well as control strains were tested for the susceptibility to defensins, killing activity of professional phagocytes and adhesion to A549 cell line. Because S. aureus are exposed to many cationic proteins in the host, we challenged a clinical isolate with minimal subinhibitory concentration (subMIC) of protamine and found that hemin and menadione auxotrophic SCVs emerged. SCVs were more resistant than normal strains to protamine but not to dermaseptin. The susceptibility to the bactericidal activity of magainin was the same for normal and SCV strains. The protamine resistance of normal as well as SCVs was strongly enhanced by high salt concentration. The adhesion of some SCVs to A549 cells was higher than adhesion of parental strains. However, the number of adherent bacteria (SCVs) was diminished in the presence of hemin for hemin auxotrophs. The uptake of SCVs by granulocytes was lower than ingestion of normal strains, but SCVs were killed with equal or greater potency. SCVs are adapted to intracellular survival and persistence in the host under certain circumstances. The ability to form a variant subpopulation affords S. aureus additional survival options.     

The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles

Intracellular replication of Salmonella enterica occurs in membrane-bound compartments, called Salmonella-containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella-pathogenicity island-2 (SPI-2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 microm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule-dependent. An intact Golgi, SseG and SPI-2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live-cell imaging between 4 and 8 h post invasion revealed that the majority of wild-type SCVs displaced less than 2 microm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 microm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi-associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.     

Identification of antimicrobial compounds active against intracellular Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus exhibit characteristics of bacteria that can penetrate mammalian cells and remain intracellular and innocuous for indefinite periods. These properties make SCVs a convenient tool that can be used to identify new antibacterial agents having activity against intracellular, quiescent bacteria. Agents active against SCVs could be useful in the treatment of chronic staphylococcal infections such as bovine mastitis. An hemB deletion mutant of S. aureus Newbould, a bovine mastitis isolate, having a stable, genetically defined SCV phenotype, was used in a screening program to identify compounds active against intracellular, gram-positive bacteria. Out of more than 260,000 compounds screened, nine compounds having the desired properties were identified. The range of MICs against gram-positive bacteria was < or = 0.12-32 microg ml-1. One of the compounds (no. 8) showed excellent activity against gram-positive (MICs < or = 0.12 microg ml-1) and gram-negative (MICs < or = 0.12-4 microg ml-1) bacteria. Each of the nine compounds demonstrated efficacy in a neutropenic mouse thigh infection model. Two compounds, including compound no. 8, reduced numbers of bacteria in a mouse mastitis model of infection. Application of a stepwise screening process has identified lead compounds that may be useful for treating persistent, intracellular infections.     

一株金黄色葡萄球菌小菌落突变株的分离鉴定

摘要：【目的】由于金黄色葡萄球菌（金葡菌）小菌落突变株（small colony variants,简称SCVs ）可引起持续复发性感染,且对氨基糖苷类有抗药性,在临床诊断和治疗上造成很大的困扰。我国国内尚无金葡菌SCVs的报道,本研究旨在分离鉴定出金葡菌SCVs菌株,为国内进行SCVs的相关研究提供生物学材料。【方法】通过细菌的形态鉴定、种特异性基因（nuc）的PCR扩增鉴定以及系列生化实验,从人源、动物源及环境源共104株金葡菌分离株中筛选得到金葡菌SCVs,并通过甲萘醌、硫胺素、胸腺嘧啶和血红素等补     

Salmonella enterica serovar Typhimurium interaction with dendritic cells: impact of the sifA gene

Salmonella enterica serovar Typhimurium (S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA, invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-gamma production compared to S. Typhimurium aroC derivatives.     

Expression of constitutively active Rab5 uncouples maturation of the Salmonella-containing vacuole from intracellular replication

The enteric bacterial pathogen Salmonella typhimurium enters and proliferates within both phagocytic and non-phagocytic host cells. Upon entry, the bacteria reside in membrane-bound vacuoles (SCVs) that mature with time, as evidenced by the sequential loss of early endosomal markers, followed by the selective recruitment of a number of lysosomal membrane glycoproteins (LAMPs). This remodelling process renders the SCVs non-fusogenic with lysosomes and is also thought to create a vacuolar environment permissive for replication. We demonstrate that disruption of the endocytic pathway by the expression of a constitutively active form of the small GTPase rab5 (rab5Q79L) significantly altered the biogenesis of the SCVs without inhibiting bacterial replication in HeLa cells. Expression of rab5Q79L caused the retention of early endosomal markers on SCVs and early acquisition of LAMP2, and led to an increase in the kinetics of intracellular replication. We also demonstrate that a significant fraction of LAMP2 in SCVs is derived from the cell surface via endocytosis rather than via the biosynthetic route. Further, in fibroblasts lacking a functional AP3 adaptor complex, in which all newly synthesized LAMP is delivered to the cell surface, recruitment of LAMP to the SCVs remained unaffected. These findings raise the possibility that all the SCV-associated LAMP could be derived by endocytosis from the cell surface.     

Searching for Molecular Players Differentially Involved in Neurotransmitter and Neuropeptide Release

Neurotransmitters and neuropeptides are stored in small clear vesicles (SCVs) and large dense core vesicles (LDCVs), respectively. Many differences in the properties of SCVs and LDCVs suggest that these two classes of secretory organelles may employ different sets of molecules in exocytosis. Relatively little is known, however, about factors that differentially participate in SCVs and LDCVs release. This article briefly overviews some key molecules that are possibly involved in the differential regulation of the trafficking, docking, priming and fusion of SCVs and LDCVs. Special issue article in honor of Dr. Ji-Sheng Han.     

A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly.

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.     

新疆动物源大肠杆菌小菌落变异体生物学特性分析

【背景】小菌落变异体(small colony variants, SCVs)在细菌耐药和持续残留等过程中发挥着重要作用，但目前国内鲜有动物源大肠杆菌(Escherichiacoli,EC)SCVs的相关报道。【目的】对新疆动物源EC-SCVs的生物学特性进行对比研究分析，为国内EC-SCVs的相关研究提供基础数据。【方法】使用氨基糖苷类抗生素对新疆动物源大肠杆菌进行诱导，使其形成SCVs。然后对野生株和诱导株进行培养特征、生化特性、药物敏感性、运动力、生物被膜和溶血活性等生物学特性检测。【结果】经卡那霉素和庆大霉素诱导，从牛、羊和马源大肠杆菌中得到5株EC-SCVs(2株为血红素依赖型，3株未知其营养依赖型)。与野生株相比，EC-SCVs的生化特性改变，所有EC-SCVs转变为不利用醋酸盐，在不同培养基中的培养特征也存在差异。并且对氨基糖苷类抗生素的耐受性增强，生物被膜形成能力增强，但运动力下降。除血红素依赖型EC-SCVs的溶血活性增强外，其他SCVs的溶血活性并无差异。【结论】动物源EC-SCVs的生物学特性与野生株相比有较大差异，这些生物学特性的改变可能会给大肠杆菌致病或耐药等...     

Augmented expression of polysaccharide intercellular adhesin in a defined Staphylococcus epidermidis mutant with the small-colony-variant phenotype

While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific beta-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.     

Characteristics of atypical forms of staphylococci (SCVs) isolated from patients with osteomyelitis

Small-colony variants (SCVs), isolated from a population of the parental strains of Staphylococcus aureus, S. haemolyticus and S. epidermidis lost a number of features typical of the species and genus and were characterized by delayed growth, altered colony morphology, lack of pigmentation and changed carbohydrate consumption. Some SCVs of S. aureus had no plasmocoagulase and lecithinase activities. The analysis of 14 SCVs showed that they were auxotrophic for hemin and menadione and resistant to aminoglycoside antibiotics. Such aberrant phenotypic characteristics complicated or made it impossible their identification by the common clinical laboratory methods. The tRNA intergenic spacer length polymorphism analysis was used to identify the atypical forms of the staphylococci.     

Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small-colony variants (SCVs) of Staphylococcus aureus represent a slow-growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild-type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin-induced SCV). Applying an ultra-high resolution chromatography and high mass accuracy MS(E) -based label-free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase-controlled manner covering early-exponential, late-exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down-regulation of the tricarboxylic acid (TCA) cycle-related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin-induced SCVs, the clinically derived SCVs showed no prominent up-regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up-regulation of TCA cycle-related parts, while oxidative stress-related proteins were down-regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain-interrupting mutants and their complemented counterparts.     

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella‐containing vacuole

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella‐containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3‐phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30–60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella‐induced filaments (Sifs) was altered by SNX3 knock‐down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.     

Accelerated s-nitrosothiol breakdown by amyotrophic lateral sclerosis mutant copper,zinc-superoxide dismutase

Mutations in copper,zinc-superoxide dismutase (SOD) have been implicated in familial amyotrophic lateral sclerosis (FALS). We have investigated the breakdown of S-nitrosothiols by wild-type (WT) SOD and two common FALS mutants, alanine-4 valine (A4V) SOD and glycine-37 arginine (G37R) SOD. In the presence of glutathione, A4V SOD and G37R SOD catalyzed S-nitrosoglutathione breakdown three times more efficiently than WT SOD. Indeed, A4V SOD catabolized GSNO more efficiently than WT SOD throughout the physiological range of GSH concentrations. Moreover, a variety of additional S-nitrosothiols were catabolized more readily by A4V SOD than by WT SOD. Initial rate data for fully reduced WT SOD and A4V SOD, and data using ascorbic acid as the reductant, suggest that FALS mutations in SOD may influence the efficiency of reduction of the copper center by glutathione. We have identified a potentially toxic gain of function of two common FALS mutations that may contribute to neurodegeneration in FALS.     

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

In epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella -containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (lgps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immunofluorescence microscopy and epitope-specific flow cytometry. We show that proteins specific to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when lgp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the lgp is acquired, and that these processes occur progressively and not as the result of a single fusion/fission event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of lgp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.     

Genetic complexity of fusidic acid-resistant small colony variants (SCV) in Staphylococcus aureus

FusE mutants are fusidic acid-resistant small colony variants (SCVs) of Staphylococcus aureus that can be selected with aminoglycosides. All FusE SCVs have mutations in rplF, encoding ribosomal protein L6. However, individual FusE mutants including some with the same mutation in rplF display auxotrophy for either hemin or menadione, suggesting that additional mutations are involved. Here we show that FusE SCVs can be divided into three genetic sub-groups and that some carry an additional mutation, in one of the genes required for hemin biosynthesis, or in one of the genes required for menadione biosynthesis. Reversion analysis and genome sequencing support the hypothesis that these combinations of mutations in the rplF, hem, and/or men genes can account for the SCV and auxotrophic phenotypes of FusE mutants.