The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Expression of Ralstonia solanacearum type III secretion system is dependent on a novel type 4 pili (T4P) assembly protein (TapV) but is T4P independent

Type IV pili (T4P) are virulence factors in various pathogenic bacteria of animals and plants that play important roles in twitching motility, swimming motility, biofilm formation, and adhesion to host cells. Here, we genetically characterized functional roles of a putative T4P assembly protein TapV (Rsc1986 in reference strain GMI1000) and its homologue Rsp0189, which shares 58% amino acid identity with TapV, in Ralstonia solanacearum. Deletion of tapV, but not rsp0189, resulted in significantly impaired twitching motility, swimming motility, and adhesion to tomato roots, which are consistent as phenotypes of the pilA mutant (a known R. solanacearum T4P-deficient mutant). However, unlike the pilA mutant, the tapV mutant produced more biofilm than the wild-type strain. Our gene expression studies revealed that TapV, but not Rsp0189, is important for expression of a type III secretion system (T3SS, a pathogenicity determinant of R. solanacearum) both in vitro and in planta, but it is T4P independent. We further revealed that TapV affected the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway, consistent with previous reports that PhcA positively regulates expression of pilA and prhG. Moreover, deletion of tapV, but not rsp0189, significantly impaired the ability to migrate into and colonize xylem vessels of host plants, but there was no alteration in intercellular proliferation of R. solanacearum in tobacco leaves, which is similar to the pilA mutant. The tapV mutant showed significantly impaired virulence in host plants. This is the first report on the impact of T4P components on the T3SS, providing novel insights into our understanding of various biological functions of T4P and the complex regulatory pathway of T3SS in R. solanacearum.     

SlyA regulates type III secretion system (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937

The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.     

Spa47 is an oligomerization‐activated type three secretion system (T3SS) ATPase from Shigella flexneri

Gram‐negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti‐infective agents.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH‐AopB‐AopD complexes in the type III secretion system of Aeromonas hydrophila

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH‐AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16–147) and the C‐terminal amphipathic helical domain (DF2, residues 242–296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N‐terminal coiled coil domain (BF1, residues 33–264). Oligomerization of the AcrH‐AopB complex is mainly through the C‐terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C‐terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.     

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.     

The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi

To ensure the optimal infectivity on contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream of hrpG, encodes a T3SS‐dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on elicitation of disease symptoms in the host and a hypersensitive response in non‐host plants, and the secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild‐type results in delayed HR and reduced t3ss expression. The results of protein–protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial cytoplasm, which is reduced by a single amino acid substitution at the 67th lysine residue of HrpF with alanine. Taken together, the data presented here suggest that HrpF has two roles in the assembly of a functional T3SS: one by acting as a negative regulator, possibly involved in the HrpSVG regulation circuit via binding to HrpG, and the other by stabilizing HrpA in the bacterial cytoplasm via HrpF–HrpA interaction prior to the secretion and formation of Hrp pilus on the bacterial surface.     

A CysB regulator positively regulates cysteine synthesis,expression of type III secretion system genes,and pathogenicity in Ralstonia solanacearum

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.     

V型分泌系统(T5SS)在禽致病性大肠杆菌中的分布及流行情况

【背景】禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)可引起禽的大肠杆菌病,严重危害养禽业。V型分泌系统(Type V secretion system,T5SS)在APEC感染过程中发挥重要作用。【目的】分析不同致病型大肠杆菌的T5SS在APEC中的分布规律,探讨T5SS与APEC的大肠杆菌进化分群及其他毒力因子的关联性。【方法】根据大肠杆菌的15个T5SS序列设计特异性引物,采用PCR检测T5SS在APEC临床分离株中的分布;分析APEC菌株的系统进化分群及毒力因子分布,探讨T5SS分布和APEC系统进化分群及毒力因子的相关性。【结果】T5SS在APEC临床分离株中广泛分布,其中ydeK和pplfP的分布率最高,分别为98.55%和92.03%;而upaC和pic的分布率均低于10%。系统进化分群结果显示,APEC主要属于A、B1和D进化分群,B2群较少;T5SS分布和进化分群分析发现ehaA、ehaB、pic、vat在D进化分群APEC菌株中分布率较高,而ehaG、ag43/flu、apaC主要分布于A及B1群APEC中。然而,T5SS和APEC其他毒力基因分布无明显的关联性。【结论】T5SS广泛存在于APEC分离株中,且部分T5SS分布与大肠杆菌系统进化分群存在关联性。     

鸭疫里默氏杆菌九型分泌系统(T9SS)分泌蛋白B739_0093的功能鉴定

【目的】鸭疫里默氏杆菌是引起鸭浆膜炎的一种革兰氏阴性病原菌,该菌编码的九型分泌系统(type IX secretion system, T9SS)参与滑动、致病等过程。前期研究结果表明,鸭疫里默氏杆菌CH-1株B739_0093基因在限铁培养条件明显上调。序列分析表明,B739_0093编码蛋白含有一个T9SS分泌蛋白保守的C端结构域,然而其具体功能未知。本研究旨在鉴定该基因编码蛋白是否被T9SS分泌,以及在该菌致病中的作用。【方法】用荧光定量PCR检测B739_0093是否被铁离子和铁转运调节子(ferric uptake regulator, Fur)调控;用大肠杆菌表达重组B739_0093截短蛋白制备多克隆抗体,通过Western blotting检测是否由T9SS分泌;构建B739_0093基因缺失株,通过毒力和定殖试验鉴定B739_0093在鸭疫里默氏杆菌致病中的功能。【结果】荧光定量PCR结果表明,B739_0093基因在铁离子限制性培养基明显上调,此调控是由调控蛋白Fur介导的;Western blotting结果显示,B739_0093基因编码蛋白在亲本株RA CH-1主要定位在分泌物中,而在T9SS缺失株定位在菌体且不能在分泌物被检测到;与亲本株RA CH-1相比,RA CH-1ΔB739_0093对雏鸭的致病力减弱,在雏鸭各组织器官的定殖能力明显降低。【结论】B739_0093基因编码蛋白是由鸭疫里默氏杆菌T9SS分泌的,其表达受铁离子及Fur调控,并且参与了该菌的致病。     

The aluminium(III)-sialic acid interaction: A potential role in aluminium-induced cellular membrane degeneration

The coordination between Al(III) and sialic acid (N-acetylneuraminic acid, HL, pK_a = 2.58 ± 0.01) was studied by potentiometric titrations at 25 °C in aqueous 0.2 M KCl, by ¹H NMR, and by electrospray ionization mass spectrometry (ESI-MS). The potentiometric measurements gave the following aluminium complex stoichiometries and stability constants: , log β(AlLH₋₂) = −6.34 ± 0.02, and log β(AlL₂H₋₁) = −1.14 ± 0.04. The ¹H NMR spectra yielded structural information on species . The ESI-MS data confirmed the metal-ligand stoichiometry of the complexes.The metal-ligand speciation at micromolar Al(III) concentrations (i.e., under in vivo conditions) at physiological pH values reveals that considerable amount of Al(III) is complexed. This suggests that the toxic effect of Al(III) towards cellular membranes might be due to its coordination by protein-bound sialic acid.     

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein''s N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

Structural characterization of the Yersinia pestis type III secretion system needle protein YscF in complex with its heterodimeric chaperone YscE/YscG

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as “chaperones” to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 Å resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonas aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.