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?     

沙门氏菌毒力岛及Ⅲ型分泌系统研究进展

沙门氏菌(Salmonella)是寄生于人和动物肠道内的革兰氏阴性杆菌,能引起人和动物多种不同临床症状表现,为人类食物中毒主要病原菌之一。沙门氏菌的侵袭力与毒力岛(pathogenicity island,PI)及其编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)直接相关。本文对沙门氏菌毒力岛、T3SS组成、T3SS分泌及调控机制、T3SS与沙门氏菌致病性、应用研究等进行综述,以期为深入研究提供参考。     

Dissecting virulence: systematic and functional analyses of a pathogenicity island

Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach. These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.     

The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition.

A large-molecular-weight capsular polysaccharide was isolated from strains of Bacteroides fragilis subspecies fragilis. By means of electron microscopy and staining with ruthenium red, the thick polysaccharide capsule was also visualized. With use of a radioactive antigen-binding assay, antibody to this capsular polysaccharide was demonstrated in antisera prepared in rabbits to each of eight strains of B. fragilis fragilis. Antibody of similar specificity was not found in antisera prepared to Bacteroides melaninogenicus or to strains of Bacteroides fragilis subspecies vulgatus and Bacteroides fragilis subspecies distasonis; such antibody was found in antisera to only one of two strains of Bacteroides fragilis subspecies thetaiotaomicron. The radioactive antigen-binding assay is a sensitive test for the detection of antibody to capsular polysaccharide. This polysaccharide antigen may form the basis of a serogrouping system for B. fragilis.     

Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF⁻ mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.     

The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes

The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P. aeruginosa genome. We have identified and characterized two P. aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several P. aeruginosa cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P. aeruginosa strains, by directing additional pathogenic functions.     

Epidemiology, antimicrobial susceptibility, pathogenicity, and significance of Bacteroides fragilis group organisms isolated at Los Angeles County-University of Southern California Medical Center

The epidemiology of species of the Bacteroides fragilis groups isolated at Los Angeles County-University of Southern California Medical Center was examined. In addition, frequency of resistance to six beta-lactam antibiotics (cefmetazole, cefotetan, ceftizoxime, imipenem, penicillin, and cefoxitin) and to clindamycin, chloramphenicol, and metronidazole was determined for each species. While B. fragilis was most commonly isolated, the other species of the B. fragilis group accounted for half of the isolates. Seven percent of 1,128 patients with infections due to species of the B. fragilis group were bacteremic. A review of bacteremic cases indicated that non-fragilis species were highly pathogenic. Resistance to clindamycin ranged from 8% to 22% among species and was most common among isolates of Bacteroides distasonis and Bacteroides thetaiotaomicron. Significant differences in antimicrobial activity were noted among the agents tested. Only imipenem, chloramphenicol, and metronidazole were predictably effective against non-fragilis species of the B. fragilis group. Prompt identification of species and susceptibility testing of clinical isolates of this group are needed if a newer beta-lactam agent or clindamycin is to be used for initial therapy.     

Metabolic activation of polycyclic aromatic hydrocarbons by human colonic mucosa and Bacteroides fragilis

Abstract The Ames Salmonella mutagenicity assay has been used to assess the metabolic activation of the following polycyclic aromatic hydrocarbons by human colonic microsomes (S9) and a cell-free extract of Bacteroides fragilis (Bf): 2-aminoanthracene, 1-naphthylamine, 2-naphthylamine, 2-aminofluorene, 2-acetylaminofluorene, anthracene, benzo(a)pyrene, 3-methylcholanthrene, 7, 12-dimethylbenzanthracene, acridine, 9-aminoacridine and 3-methylindole.
2-Aminoanthracene and 2-aminofluorene were the only compounds activated. In both cases, activation was dose-dependent and 2-aminofluorene exhibited synergistic activation by S9+Bf, as has previously been demonstrated with 2-aminoanthracene.
The organospecific aliphatic colonic carcinogen, 1, 2-dimethylhydrazine was not activated in this system. Metabolic activation by S9+Bf is thus restricted to aromatic compounds with three rings and an amino group in position 2.
These findings are consistent with enzymic substrate specificity, and are compatible with an enzymic basis for activation of polycyclic aromatic hydrocarbons by B. fragilis , present in large concentrations in the colonic lumen, and colonic microsomes.     

Determination of Helicobacter pylori virulence by analysis of the cag pathogenicity island isolated from Iranian patients

BackgroundThe cag pathogenicity island (PAI), which can be divided into two parts, cagI and cagII, is the most well-known virulence factor of Helicobacter pylori.AimsWe investigated the association between genetic variations within the cag PAI (cagA and cagE in the cagI and cagT in the cagII) and clinical outcomes in Iranian patients.SubjectsA total of 231 patients including 182 patients with gastritis, 41 with peptic ulcer and 8 with gastric cancer.MethodsThe presence of the cagA, cagE and cagT genes were measured by polymerase chain reaction and the results were compared with clinical outcomes and gastric histology.ResultsThe cagA, cagE and cagT genes were found in 154 (66.7%), 90 (39.0%) and 70 (30.3%) of clinical isolates. At least 144 (62.3%) strains possessed partially deleted cag PAI (e.g., 69 [29.9%] strains were cagA-positive, but cagE and cagT-negative).ConclusionThe single genes as well as the combination of genes in the cag PAI appeared not to be useful markers to predict H. pylori-related diseases in Iranian patients. The genomic sequences of the cag PAI in Iranian strains might be considerably different from those in other geographic locations.     

An in vivo gene deletion system for determining temporal requirement of bacterial virulence factors

Analysis of phenotypes associated with specific mutants has been instrumental in determining the roles of a bacterial gene in a biological process. However, this technique does not allow one to address whether a specific gene or gene set is necessary to maintain such a process once it has been established. In the study of microbial pathogenesis, it is important but difficult to determine the temporal requirement of essential pathogenic determinants in the entire infection cycle. Here we report a Cre/loxP-based genetic system that allowed inducible deletion of specific bacterial genes after the pathogen had been phagocytosed by host cells. Using this system, we have examined the temporal requirement of the Dot/Icm type IV protein transporter of Legionella pneumophila during infection. We found that deletion of single essential dot/icm genes did not prevent the internalized bacteria from completing one cycle of intracellular replication. Further analyses indicate that the observed phenotypes were due to the high stability of the examined Dot/Icm protein. However, postinfection deletion within 8 h of the gene coding for the Dot/Icm substrate, SdhA, abolishes intracellular bacterial growth. This result indicates that the Dot/Icm transporter is important for intracellular bacterial growth after the initial biogenesis of the vacuole. Our study has provided a technical concept for analyzing the temporal requirement of specific bacterial proteins or protein complexes in infection or development.     

Bacteroides fragilis endocarditis,bacteremia and other infections treated with oral or intravenous metronidazole

The effect on thyroid status of changing from thyroid USP to sodium L-thyroxine was evaluated in 40 patients. With thyroid, abnormally high trilodothyronine (T3) levels were seen in 36 of 38 patients receiving doses of 90 to 240 mg; compared to sodium L-thyroxine, 0.15 to 0.2 mg, the serum T3 was higher (289 ± 15 ng/dl versus 176 ± 9 ng/dl, p < 0.0005) and the thyroxine (T4) lower (7.4 ± 0.3 μg/dl versus 11.6 ± 0.5 μg/dl, P < 0.01). Thyrotoxic symptoms occurred in six patients and diminished or disappeared after the change to sodium L-thyroxine, suggesting that the raised T3 level with thyroid may have undesirable effects in some patients. The T4 level, because it is low whether symptoms are present or not, may inadvertently suggest the need for higher dosage of desiccated thyroid in patients who have already received adequate replacement. The dose of sodium L-thyroxine was adequately assessed by measurement of both T4 and T3 levels. Thyroid USP should be discontinued as thyroid medication since it produces thyroid hormone levels that are misleading estimates of thyroid function and can cause thyrotoxic symptoms.     

cag致病岛在中国人感染幽门螺杆菌中的结构特征及分类价值

目的：探讨cag致病岛基因群在中国人感染的幽门螺杆菌（Hp）中结构特征及其与临床疾病的关系,以及根据cag致病岛结构对Hp进行分类的意义。方法：合成五对针对cagA、cag致病岛中的cagⅠ、cagⅡ、cagⅠ与cagⅡ连接处、及IS605等基因片段的引物,采用PCR方法分别检测临床分离培养的107株Hp基因中,cag致病岛及相关基因结构的存在状态。结果：cag致病岛总的阳性检出率为95．3%,其中cagA、cagⅠ、cagⅡ的阳性率分别为92．5％、86．9％、69．2％,在慢性胃炎、消化性溃疡、胃癌等不同疾病组间的检出率差异无显著性（P＞0．05）。IS605的阳性检出率为43．9％,在慢性胃炎中的检出率（52．2％）明显高于十二指肠溃疡中的检出率（13．6％,P＜0．05）;cagI与cagⅡ连接处（呈连续状态存在的cag致病岛）的检出率仅为4．7％。其在十二指肠溃疡中的检出率（14．8％）明显高于慢性胃炎（1．5％）等（P＜0．01）。cag致病岛阴性的Hp菌株主要来源于慢性胃炎。cagI总的检出率（86．9％）明显高于cag Ⅱ（69．2％）,差异在慢性胃炎中有显著性（P＜0．01）;另外,cagI部分片段缺失的中国Hp菌株,其中1株为仅有cagA存在、cagⅡ及cagI其它部分均缺失的Hp的IS605阳性扩增片段明显大于国外标准菌株及其它国内阳性菌株。结论：cag致病岛在中国人群感染Hp中的存在频率极高,其结构显示出一定的地域分布特征,并在中国Hp菌株中发现新的cag致病岛结构形式存在,cag致病岛结构的变化与临床疾病的发生有一定关系,可初步将Hp划分为不同毒力的细菌群。     

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.     

Lineage-specific selection and the evolution of virulence in the Candida clade

Candida albicans is the most common cause of systemic fungal infections in humans and is considerably more virulent than its closest known relative, Candida dubliniensis. To investigate this difference, we constructed interspecies hybrids and quantified mRNA levels produced from each genome in the hybrid. This approach systematically identified expression differences in orthologous genes arising from cis-regulatory sequence changes that accumulated since the two species last shared a common ancestor, some 10 million y ago. We documented many orthologous gene-expression differences between the two species, and we pursued one striking observation: All 15 genes coding for the enzymes of glycolysis showed higher expression from the C. albicans genome than the C. dubliniensis genome in the interspecies hybrid. This pattern requires evolutionary changes to have occurred at each gene; the fact that they all act in the same direction strongly indicates lineage-specific natural selection as the underlying cause. To test whether these expression differences contribute to virulence, we created a C. dubliniensis strain in which all 15 glycolysis genes were produced at modestly elevated levels and found that this strain had significantly increased virulence in the standard mouse model of systemic infection. These results indicate that small expression differences across a deeply conserved set of metabolism enzymes can play a significant role in the evolution of virulence in fungal pathogens.

Microbial pathogens of humans typically have at least one close, nonpathogenic relative, and comparisons between the two provide a powerful entry point to identify and study disease-causing determinants. This approach has revealed numerous bacterial pathogenicity islands, clusters of genes required for a given strain or species to cause disease in humans. Because these gene clusters can be horizontally transferred, they can often be initially identified by comparing genome sequences of pathogenic and nonpathogenic strains. The situation with fungal pathogens is substantially different. Like their human hosts, these microbes are eukaryotes, and genes that work together are typically dispersed across different chromosomes rather than clustered; not surprisingly, horizontal transfer of groups of cofunctioning genes from one fungal species to another is rare. Thus, identifying and understanding how groups of genes work together to contribute to virulence remains a challenge in fungi.In this report, we consider two fungal pathogens that are closely related but differ in their virulence. Candida albicans is an opportunistic pathogen; it is a component of the normal human microbiota but is also the leading cause of systemic fungal infections in humans, which can have mortality rates as high as 40%. It is highly virulent when injected into the tail vein of mice, a standardized laboratory procedure that initiates a systemic blood stream infection. Candida dubliniensis is the closest known relative of C. albicans; it was first identified from the oral cavity of an HIV-infected patient and is typically found only as secondary infections. Although it is found throughout the world, C. dubliniensis is much less prevalent than C. albicans in the clinic; it is also less virulent in the mouse model of systemic infection, based on time-to-illness measurements (1–5).C. albicans and C. dubliniensis last shared a common ancestor nominally 10 million y ago, and their genomes are very similar in terms of gene content and synteny. A small number of individual genes are “missing” in one species compared to the other but there are also wide-scale differences in the expression of those genes conserved in both species (6–10). It has been proposed that both types of differences—in gene content and in regulation—contribute to the difference in pathogenicity, although neither idea has been directly demonstrated.In this report, we used an approach known as “allele-specific expression” to highlight patterns of gene-expression differences between C. albicans and C. dubliniensis and to identify those that bear the hallmarks of selection (11–15). Specifically, we created an interspecies hybrid between C. albicans (strain SC5314) and C. dubliniensis (strain CD36) by forcing them to mate with each other (16) (Fig. 1A). Both strains were originally isolated from human patients, and both have been studied extensively in the laboratory. In the hybrid, both parental genomes reside in the same cell, so any bias in mRNA levels must be due to cis-acting sequences specific to that genome. We show that ∼40% of orthologous gene pairs show statistically significant differences in their mRNA levels in the interspecies hybrid, although many of these differences are small in magnitude. We searched for pathways that showed a systematic up-regulation from one genome compared to the other, and were led to the genes for glycolysis, all 15 of which exhibited increased expression from the C. albicans genome compared with the C. dubliniensis genome. This pattern requires cis-acting changes to have accumulated at each of the 15 genes, all resulting in higher mRNA expression from the C. albicans alleles in the hybrid. The likelihood of this pattern emerging by chance is extremely low, and the observation strongly implies that this nonrandom pattern is the result of natural selection.Open in a separate windowFig. 1.RNA-seq of an interspecies hybrid systematically identifies gene-expression differences between C. albicans and C. dubliniensis that arose through cis-acting changes that accumulated since the two species shared a common ancestor. (A) C. albicans and C. dubliniensis (both diploid) were mated to a create tetraploid interspecies hybrid strain, using mating and auxotrophic marker complementation. We performed RNA-seq in the hybrid strain in the conditions listed, as well as the parent diploid strains in bovine plasma at 37 °C. We aligned RNA-seq reads to a concatenated C. albicans–C. dubliniensis genome and excluded reads mapping to both genomes or to multiple locations within a single genome (<5% of total reads). Systematic gene-expression differences of orthologous genes between the two species were assessed. Since both genomes were in the same trans-acting environment (that is, the hybrid strain), all measurable gene expression changes must be due to cis-acting changes. (B) Correlation between two replicate RNA-seq experiments of the interspecies hybrid grown in YPD at 30 °C. Each point represents the log₂ ratio of reads from the C. albicans genome versus reads from the C. dubliniensis genome in the interspecies hybrid, for each orthologous gene pair, with experimental replicates plotted on each axis. Points in the upper right quadrant represent genes with higher expression from the C. albicans genome than C. dubliniensis genome in the interspecies hybrid, and points in the lower left quadrant represent genes with higher expression from the C. dubliniensis genome than the C. albicans genome in that same hybrid, with Spearman correlation values shown in the lower right. We observed very high correlation between replicate experiments for all conditions tested (SI Appendix, Figs. S1 and S2).To test whether increased glycolysis gene expression could account for at least some of the virulence difference between C. albicans and C. dubliniensis, we created a C. dubliniensis strain in which the glycolysis genes were modestly overexpressed and found that this engineered strain showed enhanced virulence (compared with the parental strain) in the standard mouse model for disseminated candidiasis. This result indicates the importance of metabolic fitness in the establishment of fungal infections. More broadly, our results suggest that subtle changes in the expression of genes that are deeply conserved in all branches of life can play important roles in the evolution of specific pathogens.     

兰州市不同毒力基因型志贺菌致病力研究

目的检测2007年兰州市分离的不同毒力基因型志贺菌株致病力情况,探讨毒力基因型与致病力的关系。方法选用不同毒力基因型的志贺菌,以腹腔注射为感染途径,测定其对昆明种小鼠的半数致死量(LD50)。结果7株不同毒力基因型(分别为virA+ipaH+setlA、virA+ial+ipaH+setlA+setlB+sen、virA+setlA、virA+ial+ipaH+setlA+sen、virA+ipaH+setlA+sen、virA+ipaH+setlA+setlB、virA+ial+ipaH+setlA+setlB)志贺菌LD50分别为:1.422×109cfu/mL、6.653×107cfu/mL、6.653×108cfu/mL、1.422×109cfu/mL、1.422×109cfu/mL、6.653×108cfu/mL、3.062×109cfu/mL;结果表明,不同毒力基因型的志贺菌之间存在致病力差异,其中以基因组合为virA+ial+ipaH+setlA+setlB+sen的福氏志贺菌Ⅳ菌株的LD50最小,致病力最强。结论志贺菌的毒力基因型与致病力密切相关。     

幽门螺杆菌感染者中cag致病岛的分布及其意义

目的 探讨ｃａｇ致病岛在人幽门螺杆菌感染者中的分布特征及共与ＨＰ致的相关性。方法 采用ＰＣＲ方法检测了１０７例临床分离培养的ＨＰ菌株,分别扩增ｃａｇＡ中的２９８ｂｐ片段、ｃａｇ致病岛的ｃａｇⅠ中的１７０ｂｐ及ｃａｇⅡ中的５９７ｂｐⅡ阳性的检出率分别为２５２％和７．５％,并发现１株生胃炎,１株来源于贲门癌。ｃａｇ致病岛的检出在慢性胃炎、消化性溃疡、胃癌等疾病间差异无显著性。结论 ｃａｇ致病岛在中国人     

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2_Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2_Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2_Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2_Pto. In support of this hypothesis, HopF2_Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2_Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2_Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.