衣原体Ⅲ型分泌系统效应蛋白研究进展

Ⅲ型分泌系统（Type Ⅲ secretion system,TTSS）是一个由多组分蛋白复合体形成的跨膜通道的复杂的分子装置,它乇要通过分泌篮白,或把这些毒力蛋白直接注入宿主细胞而发挥致病作用。     

贝氏柯克斯体IV型分泌系统效应蛋白研究进展

贝氏柯克斯体是重要人兽共患病——Q热的病原,Dot/Icm IV型分泌系统(T4BSS)是其重要致病因素。贝氏柯克斯体T4BSS分泌的效应蛋白参与调控柯克斯体寄生泡发育成熟,以及宿主细胞的物质分泌运送、自噬与凋亡、转录与翻译、炎性反应等信号通路,为贝氏柯克斯体的宿主细胞内生长繁殖提供必要的场所和物质。     

铜绿假单胞菌Ⅲ型分泌系统的研究进展

铜绿假单胞菌是临床常见的重要条件致病菌,具有多种毒力因子,能引起各种急慢性感染。其中最重要的毒力因子是Ⅲ型分泌系统,主动向宿主细胞靶向输送效应蛋白,引起宿主细胞的病理变化,并逃避免疫细胞的降解。研究Ⅲ型分泌系统不仅有助于明确铜绿假单胞菌的致病机制,更重要的是为临床治疗及新药研发提供新思路。     

衣原体Ⅲ型分泌系统效应蛋白研究进展

衣原体（Chlamydia）是一类进化来源尚未明确、发育周期独特、感染宿主广泛、致病表现复杂、介于细菌和病毒之间的革兰染色阴性微生物。当有传染性无代谢活性的原体（Elementary body,EB）粘附并进入真核宿主细胞后,分化为没有传染性但有代谢活性的网状体（Reticulate body,RB）,在寄生空泡内通过二分裂复制几代后,成为包涵体。网状体生长增殖持续18-36h后又可重新分化回原体。衣原体和很多病原体一样,把分泌各种毒力蛋白作为它们的致病机制,分泌的毒力蛋白称为效应蛋白。     

金黄色葡萄球菌VII型分泌系统研究进展

细菌分泌系统是细菌依赖分泌通路进行蛋白质的跨胞浆膜转运系统,该分泌系统中存在的蛋白或蛋白性质的毒力因子与细菌的生存、繁殖和扩散紧密相关。已发现的几种分泌系统中,VII型分泌系统主要存在于革兰氏阳性菌中,其不仅与细菌的生长和致病性密切相关,而且可能在细菌毒力分泌方面发挥关键作用。本文将对金黄色葡萄球菌VII型分泌系统类型、分泌底物结构、对金黄色葡萄球菌的毒力调控机制等方面进行论述。     

沙眼衣原体III型分泌系统效应蛋白研究进展

沙眼衣原体(Chlamydia trachomatis,Ct)是专性细胞内致病菌,主要通过III型分泌系统(type III secretion system,T3SS)直接将效应蛋白分泌到宿主细胞。T3SS效应蛋白在Ct发育以及与宿主细胞相互作用中发挥重要作用,如调节宿主细胞肌动蛋白骨架、破坏免疫信号通路以及控制宿主细胞凋亡等。本文综述了沙眼衣原体 T3SS效应蛋白最新研究进展。     

志贺菌Ⅲ型分泌系统研究进展

Ⅲ型分泌系统（T3SS）是革兰阴性致病菌重要的分泌系统,细菌通过T3SS将毒力蛋白注入宿主细胞。志贺菌在与宿主肠道上皮细胞接触后,激活T3SS并将效应子蛋白注入真核宿主细胞内,引起细菌性痢疾。本文综述了志贺菌T3SS的结构与功能,从分子水平揭示了志贺菌的致病机理。     

沙门菌Ⅲ型分泌系统组成与装配研究进展

沙门菌(Salmonella)是引发人和动物食物中毒、胃肠炎的主要食源性病原菌,该菌III型分泌系统(T3SS)对其入侵宿主细胞发挥着重要作用,近年来,有关沙门菌T3SS的组成、装配以及相关致病机理的研究取得了一定进展。本文对沙门菌T3SS的组成与装配等研究作一综述,为深入研究沙门菌的致病机制以及预防、治疗该菌引发的疾病提供新的策略和手段。     

空肠弯曲菌VI型分泌系统结构特征和菌株中分布

目的 明确中国空肠弯曲菌VI型分泌系统基因簇的结构特征及其在菌株中的分布。方法 对中国不同来源空肠弯曲菌菌株进行全基因组测序,并与数据库不同来源空肠弯曲菌菌株基因组序列和VI型分泌系统基因簇做比较分析。结果 52株空肠弯曲菌经基因组系统发育分析被分为了6个进化分支,对应不同来源的菌株。空肠弯曲菌的VI型分泌系统基因簇大小约14 kb,包含14个基因,其中hcp等9个主要基因与其他细菌T6SS相同基因的同源性较高,但仍具有空肠弯曲菌独特的基因簇结构。有36.5%(19/52)空肠弯曲菌菌株含有VI型分泌系统基因簇,主要为鸡来源分支和混合来源分支中的鸡来源菌株;有83.3%(10/12)中国的鸡来源菌株携带VI型分泌系统基因簇。结论 空肠弯曲菌的VI型分泌系统与鸡来源菌株密切相关。     

布鲁氏菌的IV型分泌系统

布鲁氏菌病是由布鲁氏菌引起的一种人兽共患病,给养殖业、人类健康和动物性食品安全带来很大威胁。布鲁氏菌不产生外毒素、没有荚膜、鞭毛、质粒,不形成芽孢,缺乏这些经典的致病因子,但是该菌却具有较强的侵袭力,并且在宿主细胞内的生存繁殖能力极强,因此布鲁氏菌的相关毒力因子的研究一直是布鲁氏菌病中的重要问题之一。布鲁氏菌的IV型分泌系统在布鲁氏菌的致病力上发挥重要作用,与布鲁氏菌在宿主细胞内生存、复制有密切关系。IV型分泌系统由virB操纵子编码,由同一启动子调控,是一个多蛋白的跨膜复合物,受感染宿主细胞类型、生长温度、营养条件、PH值等多因素调控。     

A novel stabilization mechanism for the type VI secretion system sheath

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssA_S) and a long form (TssA_L). While TssA_L proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssA_S-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches.

Bacteria live in complex polymicrobial communities that are shaped by interspecies cooperation and competition. As resources are limited, antagonistic strategies are a major driver of survival and success for bacterial populations. One of the most elaborate bacterial weapons is the type VI secretion system (T6SS), which not only promotes interbacterial and interkingdom competition (1–3), but also is involved in the interaction of bacteria with their hosts (4, 5) and the acquisition of both nutrients (6, 7) and genetic material (8, 9).The T6SS apparatus is a contractile nanomachine that delivers proteinaceous effectors to neighboring cells in a contact-dependent manner (10, 11). The system is a multiprotein complex (12) that when fully assembled extends across the entire width of the cell (13). Three main components make up this structure: the membrane complex, the baseplate, and the tail, comprising the contractile sheath and the inner tube. The membrane complex (TssJLM) spans the cell envelope (14–16), providing a platform onto which the baseplate (TssEFGK) docks (17, 18). Once the baseplate is in position, it promotes the polymerization of the contractile sheath (TssBC), which encompasses the Hcp tube topped by the VgrG/PAAR complex (19–21). On sheath contraction (22) the Hcp tube and the VgrG tip, along with their associated effectors, are propelled outside the attacker and into neighboring cells (10, 11). This is followed by ClpV-dependent sheath disassembly and recycling before another round of firing (23–25). A schematic representation of a T6SS at the moment of sheath contraction is shown in Fig. 1A.Open in a separate windowFig. 1.Short TssA proteins interact with a yet undescribed class of T6SS structural components. (A) Schematic representation of a T6SS apparatus at the moment of firing. The Hcp tube (black), the VgrG/PAAR tip complex (gray/black), and their associated effectors (yellow) are propelled from the attacking cell into a neighboring target cell (purple). The membrane complex and baseplate structures are shown in gray, while the sheath and the cap protein TssA are depicted in blue and red, respectively. (B) Synopsis of the results from the in silico analysis presented in Dataset S1. Average protein sizes in kDa are given for T6SS components from each phylogenetic group, and the domain architecture of TssA-like proteins is shown; the N terminus of all TssA-like proteins forms an ImpA domain, while their C terminus varies (28, 29). “TagA” refers to homologs of the E. coli TagA anchor protein (30), “TagA_V” has been described as the anchor for the T6SS of V. cholerae (29), and “TagA_O”, which has a different C terminus than the other two forms of TagA, has not been described previously. Representative examples of well-studied T6SSs from each phylogenetic group are given in the footnote. (C) Dot blot assays showing interaction of P. putida TagB1 with itself and its cognate TssA1. CcmG-StrepII was used as a binding control protein; similar amounts of pure TagB1-StrepII and CcmG-StrepII were spotted on the membrane. (D–G) P. putida K1-T6SS sheaths display characteristic T6SS behaviors. (D) In vivo imaging of P. putida rpoN TssB1-sfGFP showing the full cycle of T6SS assembly over ∼120 s for a representative sheath (white arrowhead). The panels are selected images from a fluorescence microscopy time-lapse recording of P. putida rpoN expressing TssB1-sfGFP from the native tssB1 locus (Movie S1); images were recorded every 2 s. (E) Formation of P. putida sheaths is dependent on TssA1. Shown are representative fluorescence microscopy images of P. putida rpoN and P. putida rpoN tssA1 expressing TssB1-sfGFP from the native tssB1 locus. (F) Quantification of images in E. Approximately 10% of cells form sheaths in P. putida rpoN, while no sheaths are detected in the isogenic tssA1 mutant. n indicates the number of cells included in the analysis. (G) P. putida TssA1 localizes to the site of sheath initiation and subsequently migrates at the end of the polymerizing sheath. The white arrow indicates the direction of sheath extension. The panels are selected images from a fluorescence microscopy time-lapse recording of P. putida rpoN expressing sfGFP-TssA1 and TssB1-mScarlet-I from the native gene loci; images were recorded every 2 s. (Scale bars in D, E, and G: 1 μm.)TssA is a core structural component essential for T6SS function (26, 27). TssA promotes priming and polymerization of the sheath, whereby after recruiting the remaining baseplate proteins, it is displaced by the growing sheath and migrates at its distal end, eventually reaching the other side of the cell (27). Because of its central role, TssA is found in all T6SSs; nonetheless, it is not very well conserved. Notably, TssA exists in two major forms, a long 55- to 60- kDa protein (TssA_L) and a shorter ∼40 kDa version (TssA_S) (28, 29) (Fig. 1B). The structure and function of prototypical TssA_L proteins have been extensively studied (27–29), and the Escherichia coli, Vibrio cholerae, and Aeromonas hydrophila TssA_L are known to form a ring-like structure, where the C terminus of the protein largely occupies the center of the ring (27–29). It has been recently shown that these long TssA forms interact with an accessory TssA-like protein, called TagA, which secures the distal end of the sheath at the opposing cell membrane. This ensures optimal sheath polymerization and allows the sheath to be maintained in its extended conformation for long periods (29, 30). In contrast, Pseudomonas aeruginosa and Burkholderia cenocepacia TssA_S, while still ring-like, display structures in which the center of the ring is empty (26, 28). In this case, TagA is not present, and it is unknown how the sheath is stabilized and anchored. To understand the implications of TssA diversity for T6SS assembly dynamics and function, it is necessary to elucidate the relationship among short TssA proteins, the baseplate, and the extending sheath and to determine how TssA_S-containing T6SSs are stabilized.     

Type VI secretion system sheaths as nanoparticles for antigen display

The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between cells by a mechanism analogous to phage tail contraction. T6SS sheaths are cytoplasmic tubular structures composed of stable VipA-VipB (named for ClpV-interacting protein A and B) heterodimers. Here, the structure of the VipA/B sheath was exploited to generate immunogenic multivalent particles for vaccine delivery. Sheaths composed of VipB and VipA fused to an antigen of interest were purified from Vibrio cholerae or Escherichia coli and used for immunization. Sheaths displaying heterologous antigens generated better immune responses against the antigen and different IgG subclasses compared with soluble antigen alone. Moreover, antigen-specific antibodies raised against sheaths presenting Neisseria meningitidis factor H binding protein (fHbp) antigen were functional in a serum bactericidal assay. Our results demonstrate that multivalent nanoparticles based on the T6SS sheath represent a versatile scaffold for vaccine applications.The bacterial type 6 secretion system (T6SS) is a dynamic apparatus that translocates proteins between effector cells and target cells (1–4). It is conserved in 25% of Gram-negative bacteria, including Vibrio cholerae, Pseudomonas aeruginosa and Escherichia coli. The T6SS plays a crucial role in bacterial pathogenicity and symbiosis, targeting either eukaryotic cells or competitor bacterial cells (5). The assembled and functional T6SS apparatus has structural homology to bacteriophage T4 phage tail components and can be divided into two distinct assemblies: a contractile phage tail-like structure and a transmembrane complex (6). V. cholerae VipA and VipB (named for ClpV-interacting protein A and B) and orthologous proteins in other bacteria build within the cytosol of effector cells a tubular sheath structure that is anchored to the various layers of the cell envelope through its association with the T6SS transmembrane complex (7).VipA/B sheaths are composed of six protofilaments arranged as a right-handed six-start helix similar to early T4 tail sheaths (8). Each protofilament is formed by a VipA/B heterodimer, and the atomic-resolution structure of a native contracted V. cholerae sheath has been recently determined by cryo-electron microscopy (9). Stable expression of VipB in V. cholerae requires the presence of VipA, and VipA/B heterodimers can be recruited into assembled tubular sheath structures spontaneously (10, 11). Because both ends of VipA are exposed on the external surface of the sheath tubules, a C-terminal fusion of VipA protein with superfolded green fluorescent protein (sfGFP) is functional in T6SS sheath assembly and activity, as previously demonstrated (3).Because these tubular structures are assembled in cytoplasm and can be purified from bacteria (3), we explored the possibility that T6SS sheaths could be used as a new particle-based delivery system for vaccine antigens. It is thought that particulate structures used for vaccine formulations are efficiently targeted for uptake by antigen-presenting cells (APCs) and interact directly with antigen-specific B cells generating humoral responses (12). Although particulate protein antigens may be more resistant to degradation, they are eventually proteolytically processed, and the resulting peptides are presented by the major histocompatibility complex (MHC) class I and class II molecules in a process that leads to activation of CD4⁺ and CD8⁺ T-cell helper and effector responses. Examples of particulate vaccine delivery systems include lipid-based systems [emulsions, immune-stimulating complexes (ISCOMs), liposomes, virosomes], polymer-based structures (e.g., nano-/microparticles), and virus-like particles (VLPs), with each of these systems presenting their own spectrum of advantages and disadvantages for practical use as human immunogens (13).In this work, VipA/B sheaths displaying heterologous protein antigens on the surface were generated and tested as a particulate vaccine antigen delivery system. Our results show that sheath-like structures displaying different antigens were immunogenic and that antibodies elicited against one of these, the Neisseria meningitidis factor H binding protein (fHbp), were functional in a serum bactericidal assay. The T6SS antigen delivery system demonstrates potential as a multivalent particle to deliver one or more antigens simultaneously into the same antigen-presenting cell. Moreover, the use of heterologous VipA and VipB sheaths displaying a common antigen in sequential vaccine booster regimens minimizes immune responses against the delivery system itself and focuses the immune responses against the common antigen of interest.     

The Vibrio cholerae type VI secretion system displays antimicrobial properties

The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI secretion system (T6SS), which is structurally similar to the bacteriophage cell-puncturing device, has been recently identified in V. cholerae and is used by this organism to confer virulence toward phagocytic eukaryotes, such as J774 murine macrophages and Dictyostelium discoideum. We tested the interbacterial virulence of V. cholerae strain V52, an O37 serogroup with a constitutively active T6SS. V52 was found to be highly virulent toward multiple Gram-negative bacteria, including Escherichia coli and Salmonella Typhimurium, and caused up to a 100,000-fold reduction in E. coli survival. Because the T6SS-deficient mutants V52ΔvasK and V52ΔvasH showed toxicity defects that could be complemented, virulence displayed by V. cholerae depends on a functional T6SS. V. cholerae V52 and strains of the O1 serogroup were resistant to V52, suggesting that V. cholerae has acquired immunity independently of its serogroup. We hypothesize that the T6SS, in addition to targeting eukaryotic host cells, confers toxicity toward other bacteria, providing a means of interspecies competition to enhance environmental survival. Thus, the V. cholerae T6SS may enhance the survival of V. cholerae in its aquatic ecosystem during the transmission of cholera and between epidemics.     

Effects of the new oral hypoglycaemic agent nateglinide on insulin secretion in Type 2 diabetes mellitus.

AIMS: The new non-sulphonylurea oral hypoglycaemic agent nateglinide has been shown to enhance insulin secretion in animals and in healthy human volunteers and thus offers a potential advance in the treatment of Type 2 diabetes mellitus. This study examined whether nateglinide can enhance insulin secretion, and particularly the first phase insulin response, in patients with Type 2 diabetes. METHODS: A double-blind, placebo-controlled trial, examining the effects of a single oral dose of 60 mg nateglinide, given 20 min prior to an intravenous glucose tolerance test (IGTT), on insulin secretion in 10 otherwise healthy Caucasian men with recently diagnosed Type 2 diabetes (duration since diagnosis 0-44 months). RESULTS: Insulin secretion (both overall and first phase) was significantly increased by nateglinide (P < 0.001), as were C-peptide (P < 0.001) and proinsulin (P < 0.001) secretion. Overall glucose concentrations following glucose challenge were lower after nateglinide than after placebo (P = 0.05). CONCLUSIONS: Nateglinide significantly increases insulin secretion in Type 2 diabetic patients, in particular restoring the first phase insulin response. Further study is necessary to determine the effects of chronic administration on insulin secretion and blood glucose concentration.     

Incretins, insulin secretion and Type 2 diabetes mellitus

When glucose is taken orally, insulin secretion is stimulated much more than it is when glucose is infused intravenously so as to result in similar glucose concentrations. This effect, which is called the incretin effect and is estimated to be responsible for 50 to 70% of the insulin response to glucose, is caused mainly by the two intestinal insulin-stimulating hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Their contributions have been confirmed in mimicry experiments, in experiments with antagonists of their actions, and in experiments where the genes encoding their receptors have been deleted. In patients with Type 2 diabetes, the incretin effect is either greatly impaired or absent, and it is assumed that this could contribute to the inability of these patients to adjust their insulin secretion to their needs. In studies of the mechanism of the impaired incretin effect in Type 2 diabetic patients, it has been found that the secretion of GIP is generally normal, whereas the secretion of GLP-1 is reduced, presumably as a consequence of the diabetic state. It might be of even greater importance that the effect of GLP-1 is preserved whereas the effect of GIP is severely impaired. The impaired GIP effect seems to have a genetic background, but could be aggravated by the diabetic state. The preserved effect of GLP-1 has inspired attempts to treat Type 2 diabetes with GLP-1 or analogues thereof, and intravenous GLP-1 administration has been shown to be able to near-normalize both fasting and postprandial glycaemic concentrations in the patients, perhaps because the treatment compensates for both the impaired secretion of GLP-1 and the impaired action of GIP. Several GLP-1 analogues are currently in clinical development and the reported results are, so far, encouraging.Abbreviations GLP-1 Glucagon-like peptide-1 - GIP glucose-dependent insulinotropic polypeptide - FPG fasting plasma glucose     

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.Bacteria that live in communities have numerous mechanisms to compete with other strains and species. The ability to acquire nutrients is a major factor dictating the success of a species in a community. In addition, the production of secreted factors, such as bacteriocins, that competitively interfere or antagonize other strains/species, also contributes to a member’s fitness in a community. In the microbe-dense human gut ecosystem, such factors and mechanisms of antagonism by predominant members are just beginning to be described, as are models predicting the relevance of these competitive interactions to the microbial community (1). Bacteroidales is the most abundant order of bacteria in the human colonic microbiota, and also the most temporally stable (2). The fact that numerous gut Bacteroidales species stably cocolonize the human gut at high density raises the question of how these related species and strains interact with each other to promote or limit each other’s growth. We previously showed that coresident Bacteroidales strains intimately interact with each other and exchange large amounts of DNA (3) and also cooperate in the utilization of dietary polysaccharides (4). To date, two types of antagonistic factors/systems have been shown to be produced by human gut Bacteroidales species: secreted antimicrobial proteins (5) and T6SSs (3, 6, 7). However, neither of these antagonistic processes has been analyzed to determine if they provide a competitive advantage in the mammalian intestine.Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems used by some Gram-negative bacteria to intoxicate other bacteria or eukaryotic cells. The T6 apparatus is a multiprotein, cell envelope spanning complex comprised of core Tss proteins. A key component of the machinery is a needle-like structure, similar to the T4 contractile bacteriophage tail, which is assembled in the cytoplasm where it is loaded with toxic effectors (8–10). Contraction of the sheath surrounding the needle apparatus drives expulsion of the needle from the cell, delivering the needle and associated effectors either into the supernatant of in vitro grown bacteria, or across the membrane of prey cells. Identified T6SS effectors include cell wall degrading enzymes (11), proteins that affect cell membranes such as phospholipases (12) and pore-forming toxins (13, 14), proteins that degrade NAD(P)+ (15), and nucleases (16). The effector protein is produced with a cognate immunity protein, typically encoded by the adjacent downstream gene (17), which protects the producing cell from the toxicity of the effector. Although both eukaryotic and bacterial cells are targeted by T6SS effectors (18), most described T6SSs target Gram-negative bacteria.We previously performed a comprehensive analysis of all sequenced human gut Bacteroidales stains and found that more than half contain T6SS loci (7). These T6SSs are similar to the well-described T6SSs of Proteobacteria in that remote orthologs of many Proteobacterial Tss proteins are encoded by Bacteroidales T6SS regions, with the exception of proteins that likely comprise the transmembrane complex, which are distinct. The T6SS loci of human gut Bacteroidales species segregate into three distinct genetic architectures (GA), designated GA1, GA2, and GA3, each with highly identical segments within a GA comprising the core tss genes (7). GA1 and GA2 T6SS loci are present on large ∼80- to 120-kb integrative conjugative elements (ICE) that are extremely similar at the DNA level within a GA. Due to the ability of these T6SS regions to be transferred between strains via ICE, GA1 and GA2 T6SS loci are present in diverse human gut Bacteroidales species. GA3 T6SS loci are confined to Bacteroides fragilis and are not contained on conserved ICE (7).Although T6SS loci of a particular GA are highly identical to each other, each GA has internal regions of variability where the genes differ between strains (7). The variable regions of GA1 and GA2 T6SS loci contain genes encoding the identifiable toxic effector and cognate immunity proteins found in these regions. Unlike the GA1 and GA2 T6SS loci, there are no identifiable genes encoding toxin or immunity proteins in the two variable regions or other areas of GA3 T6SS loci. The present study was designed to answer three fundamental questions regarding GA3 T6SS loci: (i) Because no known effectors/immunity proteins are encoded by these regions, are they involved in bacterial antagonism? And if so, what prey cells do they target? (ii) Do the variable regions contain genes encoding effector and immunity proteins? and (iii) If GA3 T6SSs mediate bacterial antagonism, do they provide a competitive advantage in the mammalian gut?     

Type III secretion system effector proteins are mechanically labile

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (F_unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx^‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (F_unfold > 80 pN) and brittle (Δx^‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Type III secretion systems (T3SS) are large nanomachines utilized by both pathogenic and symbiotic bacteria to inject effector proteins directly into the cytoplasm of host cells (1–3). Once delivered, effector proteins facilitate host cell colonization through a variety of mechanisms (4–7), including down-regulation of the host immune response (8) and rearrangement of the cytoskeleton (9, 10). The T3SS apparatus, known as the injectisome, is a syringe-like structure with a hollow needle that spans the inner and outer bacterial membranes, the extracellular space, and the host membrane, enabling proteins to pass directly from bacteria to host cells (Fig. 1A) (2). Specialized bacterial chaperones often bind the N-terminal 50 to 100 amino acids (aa) of the effector proteins, known as the chaperone binding domain, and help maintain the effector N-terminal domain in an extended conformation. C-terminal to the chaperone binding domain, effector proteins contain one or more globular domains, which adopt their folded conformations even when in complex with their cognate chaperone (4, 11, 12). The effector proteins, or their chaperone complexes, are recognized by the base of the injectisome prior to secretion (13). At its narrowest point, the injectisome needle’s inner diameter is less than 2 nm (14–16). As a result, effector proteins must be mostly unfolded to be secreted (17–20). Secretion is thus thought to proceed by a “threading-the-needle mechanism,” where the N-terminal extended domain is released from the chaperone and fed to the injectisome, followed by unfolding of the C-terminal effector domain (21).Open in a separate windowFig. 1.Thermodynamic stability of T3SS effector proteins SptP_CD and SopE2_CD. (A) Schematic depiction of protein transport through the T3SS showing effector proteins, which are at least partially folded in the bacterial cytoplasm. Such effector proteins interact with an associated unfoldase to passage through the T3SS, which has an inner channel with a diameter <2 nm. Once inside the host cytoplasm, effector proteins refold to carry out their function. (B) Crystal structures of SptP_CD (Protein Data Bank [PDB] ID code 1G4U) and SopE2_CD (PDB ID code 1R9K). (C) Ellipticity from CD at λ = 222 nm plotted as a function of urea concentrations for SptP_CD (orange) and SopE2_CD (green). A fit of the data with Eq. 1 yielded the free energy of unfolding ΔGunfold0 for SptP_CD (6.9 ± 0.2 kcal/mol [mean ± fit error]) and SopE2_CD (6.0 ± 0.2 kcal/mol [mean ± fit error]). Data points are the result of at least three independent measurements. Error bars represent SD.Before proteins are secreted through the T3SS, they interact with a hexameric ATPase at the base of the T3SS that is capable of mediating chaperone release from effector proteins and effector-protein unfolding (15, 22). Indeed, most in vivo unfolding is catalyzed by unfoldases that work from one end of the substrate protein in stark contrast to the global effects of temperature, pH, or chemical denaturants. The most common examples of targeted protein unfolding are catalyzed by ATPases of the AAA(+) family that mechanically unfold their substrates (23, 24). For example, the AAA(+) ATPase ClpX forms a ring-shaped hexamer that mechanically pulls its substrates through its narrow central pore to unfold them (25). These are powerful unfoldases that can unfold even tightly packed proteins such as GFP, ubiquitin, and dihydrofolate reductase (DHFR) (23, 24, 26, 27). However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F₁F₀ ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15, 28–30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20, 22, 31, 32). These observations have led to the current model that T3SS effector proteins have low thermodynamic stability to facilitate their secretion (22, 31–33).While thermodynamic stability is the most common metric of protein stability, mechanical stability is a distinct metric that quantifies how easily a protein unfolds under force (F_unfold). Mechanical stability is typically measured by pulling across the N and C termini of single molecules via force spectroscopy using optical tweezers (34, 35) or an atomic force microscope (AFM) (36). Early force spectroscopy studies showed that thermodynamic stability does not correlate with mechanical stability (37–41). For example, titin’s I28 domain requires ∼20% more force to unfold than titin’s I27 domain [I85 and I91, respectively, in the new nomenclature (42)], despite I27 having approximately twofold higher thermodynamic stability (43). Importantly, AFM studies have shown that GFP (44), ubiquitin (45), and DHFR (46) are mechanically robust, requiring high forces to unfold despite their typical thermodynamic stabilities. These three proteins each stall the T3SS; thus, mechanical stability may be the physical determinant to proteins being secreted by the T3SS, rather than thermodynamic stability.Here, we determine the thermodynamic and mechanical stabilities of SptP and SopE2, two effector proteins from Salmonella enterica. These effectors are ideal candidates for this study as they have known crystal structures (10, 47), have characterized in vivo secretion kinetics (48), and represent effector proteins of different size and structure (Fig. 1B). We show that the catalytic domains of SptP and SopE2 have unremarkable thermodynamic stabilities, similar to many other previously characterized proteins, including GFP, ubiquitin, and DHFR. Conversely, our AFM-based force spectroscopy measurements demonstrate that SptP and SopE2 are among the most mechanically labile proteins studied to date by AFM. These two T3SS effector proteins are therefore mechanically labile while being thermodynamically stable, supporting the hypothesis that it is mechanical stability, not thermodynamic stability, that predicts efficient protein secretion by the T3SS.     

Progress in diabetes research in China

The prevalence of diabetes, especially Type 2 diabetes mellitus (T2DM), is increasing markedly throughout the world, including in China. Because T2DM and its complications are associated with considerable socioeconomic burden and mortality, there is increasing interest in developing strategies to prevent or delay progression of the disease. In recent decades, many researchers have focused on the mechanism of onset of diabetes, as well as examining the benefits of various interventions in subjects with different glucose tolerance status to prevent or delay development of the disease. In the present article, we focus on five areas (epidemiology, early intervention, insulin sensitivity and β-cell function, adipocytokines, and traditional Chinese medicine) to review the progress of research into diabetes in China today. The prevalence of diabetes in China is one of the highest in the world. However, with lifestyle interventions and appropriate pharmacological therapies (including traditional Chinese medicine), T2DM may be prevented, well controlled, or even put into remission. Accurate estimation of insulin secretion and insulin sensitivity, as well as better characterization of the physiological function of adipocytokines, could give us a better understanding of the basic mechanisms underlying the onset of diabetes and could lead to better interventions in people with impaired glucose tolerance and T2DM.     

Relationship of non-alcoholic hepatic steatosis to cortisol secretion in diet-controlled Type 2 diabetic patients.

AIMS: To examine the association of non-alcoholic hepatic steatosis (HS) with the activity of the hypothalamo-pituitary-adrenal (HPA) axis in Type 2 diabetic individuals. METHODS: The activity of the HPA axis, as measured by 24-h urinary free cortisol (UFC) excretion and serum cortisol levels after 1.0 mg dexamethasone, was measured in 40 diet-controlled, predominantly overweight, Type 2 diabetic patients with non-alcoholic HS and in 40 diabetic patients without HS who were comparable for age, sex and body mass index (BMI). RESULTS: Subjects with non-alcoholic HS had significantly higher 24-h UFC excretion (191 +/- 4 vs. 102 +/- 3 nmol/24 h; P < 0.001) and post-dexamethasone cortisol concentrations (29.1 +/- 2 vs. 14.4 +/- 1 nmol/l; P < 0.001) than those without HS. Patients with HS had significantly higher values for HOMA insulin resistance score, plasma triglycerides and liver enzymes. Age, sex, BMI, waist-hip ratio (WHR), diabetes duration, HbA1c, LDL-cholesterol and blood pressure values were not different between the groups. The differences in urinary and serum cortisol concentrations between the groups remained significant after adjustment for age, sex, BMI, WHR, HOMA insulin resistance score, plasma triglycerides, HbA1c and liver enzymes. In multiple logistic regression analyses, 24-h UFC or serum cortisol concentrations (P < 0.05 and P = 0.02, respectively), along with age and HOMA insulin resistance, predicted the presence of HS, independently of potential confounders. CONCLUSIONS: These results demonstrate that non-alcoholic HS is closely associated with a subtle, chronic overactivity of the HPA axis in diet-controlled Type 2 diabetic individuals.