1. Pathogenicity Island，PAI 毒力岛

2. 概念和特征 分类 功能
PAI与细菌致病性
PAI与细菌进化
PAI研究的方法和意义
几种重要细菌的PAI

3. Concept Uropathogenic E. coli. (UPEC) Goebel ‘hemolysin determinant’

4. genomic Islands (GEIs)
Concept (Hacker,1997)
genomic Islands (GEIs)
If the GEIs contain virulence genes they are referred to as pathogenicity islands (PAIs).
pathogenicity Islands (PAIs)
Encode genes involved in particular adaptive functions of medical and environmental interest via horizontal gene transfer.
virulence genes
antibiotic resistance genes
Other functional genes

6. 1997， Hacker：一个分子量很大的（>30kb）、载有多个毒力基因的不稳定的染色体DNA片段，外缘与tRNA基因和/或IS相连，存在于强毒株，在相关菌的弱毒株或无毒株中不存在或仅散在分布。

7. Characterization OF PAI
PAI carry one or more virulence genes;
Genomic elements with characteristics similar to PAI but lacking virulence genes are referred to as genomic or metabolic islands.

8. Characterization OF PAI
PAI are present in the genomes of a pathogenic bacterium but absent from the genomes of a nonpathogenic representative of the same species or a closely related species.
PAI occupy relatively large genomic regions. The majority of PAI are in the range of 5 to 200 kb.

9. Characterization OF PAI
PAI often differ from the core genome in their GC content and also show a different codon usage. The average GC content of bacterial DNA can range from 25 to 75%. Most pathogenic bacterial species have GC contents between 40 and 60%.

10. Characterization OF PAI
tRNA genes represent hot spots for the integration of PAIs. The 3′ end of tRNA genes is frequently identical to the attachment sites of bacteriophages, and therefore function as preferred target sites for integration.

11. Characterization OF PAI
PAI are frequently associated with mobile genetic elements. They are often flanked by direct repeats (DR). DR are defined as DNA sequences of 16 to 20 bp (up to 130 bp) with a perfect or nearly perfect sequence repetition.
DR act as recognition sequences for enzymes involved in excision of mobile genetic elements, thus contributing to the instability of a PAI flanked by DR.

12. Characterization OF PAI
PAI often carry cryptic or even functional mobility genes such as integrases or transposases.
PAI often are unstable and delete with distinct frequencies. Virulence functions encoded by certain PAI are lost with a frequency that is higher than the normal rate of mutation.
DR act as recognition sequences for enzymes involved in excision of mobile genetic elements, thus contributing to the instability of a PAI flanked by DR.

13. 毒力岛与毒力基因是紧密联系的两个不同概念:
毒力岛首先是基因组岛，有很多证据表明它是来源于细菌基因的水平转移。毒力岛是个群体概念，是对基因群体的研究；而毒力基因则是个体概念，着眼于单个基因。
其次，毒力岛携带有对宿主产生致病性的基因群。而从分布上说，组成毒力岛的基因群在细菌染色体的是连续的，它们的缺失（或者转移）也将是同时发生的；从功能上说，这些基因能够相互作用，而毒力的表达是这些基因群相互作用的结果。例如 Salmonella 的 SPI2编码的III型分泌系统。

14. 根据其G+C百分比与宿主菌的差异 Classification 高G+C PAI，如小肠结肠炎耶尔森菌的PAI

15. Identification of PAIs Bioinformatics method (computationally identify genomic islands in sequenced genomes.)
Identification of genomic regions that contain atypical sequence composition in comparison to the genome-wide mean, such as abnormal G + C content, dinucleotide bias, and distinct codon usage.
Advantages: Requires the analysis of a single genome (i.e. multiple reference genomes are not required); Not dependent on phylogenetic tree inference
Disadvantages: Does not detect islands if donor and recipient organisms have the same genome composition; Amelioration of genomic islands over time (such that they have the same sequence composition as the genome) restricts prediction to only relatively recent events.

16. Identification of PAIs Bioinformatics method (computationally identify genomic islands in sequenced genomes.)
Search genes with functions that are often associated with horizontal gene transfer (HGT) events, such as mobility genes (integrases, transposases, phage genes, genes with unusual similarity to phylogenetically distant species)
Advantages: Considered strong indicators of horizontal gene transfer (HGT) when combined with other methods like atypical genome composition
Disadvantages: Depends on accurate gene annotation; Only a limited set of features are known to be associated with GIs; Not associated with all Gis.

17. Identification of PAIs Bioinformatics method (computationally identify genomic islands in sequenced genomes.)
tRNA genes screening
Salmonella enterica serovars Typhimurium, Typhi (Hansen-Wester and Hensel, 2002)
four E. coli and Shigella strains (Ou et al.,2006).

18. Identification of PAIs Bioinformatics method (computationally identify genomic islands in sequenced genomes.)
Direct comparative genomics of evolutionary related species or serovars and the identification of unique regions.
Advantages: Ability to potentially detect recent and ancient HGT events; Can detect HGT between species with the same genome sequence compositions; Utilizes evolutionary history and therefore can make strong predictions
Disadvantages: Currently not very automated; Depends on methods that are often computationally intensive; Requires multiple genomic sequences

19. IslandPath
Figure. An example of part of an IslandPath for Helicobacter pylori
A yellow circle indicates its %G+C value is bigger than the high value. A light blue circle indicates its %G+C value is bigger than the low value and smaller than the high value. A pink circle indicates its %G+C value is smaller than the low value. A black line indicates a transfer RNA gene lies between the two ORFs. A purple line indicates a ribosomal RNA gene lies between the two ORFs. A deep blue line indicates both a tRNA and rRNA gene lie between the two ORFs. A black square indicates the dot is annotated as a transposase. A black triangle indicates the dot is annotated as an integrase. A strike-line across the circles represents regions with dinucleotide bias > 1 S.D.

20. Function of PAI 虽然都是编码毒力相关基因的DNA大片段，但其产物却 因不同的细菌而有差异。
具有编码III型分泌系统的基因，对致病菌侵袭宿主上皮 细胞以及在巨噬细胞内的存活具有重要意义。
编码一些表面蛋白(如菌毛、环境感受器等)和外毒素 （如hemolysin、TSST-1等）。
含有调控成分，使其在赋予宿主细菌一些新的毒力特征 的同时，还调控着其他毒力因子的功能。
同一病原菌上的两个毒力岛之间也存在着基因的互相调 控。

21. Virulence factors encoded by PAI

22. PAI

25. Function of PAI
毒力岛不仅赋予病原菌特殊的致病能力，介导感染过程的特殊阶段，而且在细菌进化过程中扮演重要角色，毒力岛的获得可能与新现病原菌密切相关。因此，毒力岛的研究为深入了解细菌的致病性、毒力因子和进化提供了有效的途径。

26. PAI与细菌进化
PAI的形成
PAI的边缘序列
PAI与细菌对环境的适应性

27. PAI的形成 基因进化: 点突变，基因重排，缺失，插入。 水平转移机制：质粒，噬菌体，转座子，IS，基因岛重组。
猜测是由质粒或噬菌体整合到染色体形成。

28. Acquisition of PAIs
Model of the development of PAI of pathogenic E. coli. In this basic model, foreign DNA is acquired by an ancient nonpathogenic E. coli strain, e.g., a normal inhabitant of the gut of vertebrates. In EHEC, a virulence-associated plasmid and at least one Stx-converting phage and several PAI have been acquired and maintained due to the specific adaptation to different environments.
Genomic islands, which are present in a specific live environment may specialize and are involved in the development of disease such as diarrhea and hemolytic-uremic syndrome after colonization of the large intestine (A), watery diarrhea after colonization of the small intestine (B), and UTI after survival and colonization of E. coli in the bladder (C). Such events probably have led to the development of specific pathotypes of E. coli, examples of which are EHEC (A), EPEC (B), and UPEC (C). In the model described here, the evolutionary sequence of uptake and incorporation of mobile genetic elements has not been considered. tRNA genes and bacterial phage attachment sites are depicted by grey rectangles with dots and hatched dark grey rectangles,respectively. stx, Shiga toxin gene; OI, O-island; espC, E. coli secreted protease gene.

29. Acquisition of PAIs
Horizontal gene transfer (HGT) is defined as the transfer of genetic material between bacterial cells uncoupled with cell division
Natural transformation: Transformation is the process whereby prokaryotes take up free DNA from their surroundings.
Conjugation: involves transfer of conjugative plasmids through a designated tube-like structure (a pilus, which represents a subset of the T4SS family) from a donor bacterium to a recipient cell that can be distantly related prokaryotic species.
Transduction : Transduction is the movement of genes from one prokaryotic species to another via viruses.

30. Different stages in the evolution of a PAI:
Acquisition of PAIs
Different stages in the evolution of a PAI:
A genetic element is acquired by a bacterial cell, through a horizontal gene transfer(transduction in this case) from an environmental flexible gene pool (1). Following a successful uptake (2), recombination which, is mediatedby an integrase (int) or other mechanisms (3) results in integration of the acquired element into the chromosome (4). Loss of the genes that are involved in the mobility of the element leads to stably integrated PAI in the chromosome (5). If the incorporated PAI confers an advantage to the organism in the specific niche, a positive selection will operate to favour this variant. Subsequently, the frequency of this variant will increase in the population over time (6). Genetic rearrangements or new gene acquisitions will lead to further evolvement of the PAI. Now the modified PAI can be recombined with the environmental available gene pool and be further transferred to a new recipient microorganism (7).

31. PAI的边缘序列 同向重复序列（DR）作用：特异性重组的靶位 点，并促进重排。 插入元件（IS）作用：与PAI的切除和整合有关
tRNA基因:许多PAI位于tRNA基因附近；tRNA 基因是外源DNA和噬菌体整合到细菌染色体的 靶位；tRNA位点在致病菌进化中起关键作用； 从tRNA基因位点寻找新的PAI。

32. PAI与细菌对环境的适应性 致病菌获得PAI，失去低选择值基因片段，使细菌更加适应外界环境 某些PAI缺失频率为10－5～10－4
链霉菌可缺失800kb的DNA，基因缺失变异株比野生株适应环境能力更强

33. 几种重要细菌的PAI

34. Salmonella pathogenicity island
SPI
Most virulence factors of S. enterica are determined by chromosomal genes, and many of these are located within PAI. The PAI of S. enterica serotype Typhimurium are referred to as Salmonella pathogenicity islands (SPI)
目前已在沙门菌中发现至少14个PAI：SPI1-SPI14。 SPI G+C mol为37～47％（染色体为52％）

35. Salmonella Structure of SPI1
SPI-1 is about 40 kb long and forms a Salmonella-specific insertion between genes that are consecutive in E. coli K-12. This PAI is not associated with a tRNA gene, and the base composition of SPI-1 is 47% G+C, lower than the average G+C content of the core genome (which is about 52%). Genetic elements involved in DNA mobility are not obvious in SPI-1, and the locus appears to be stable in all clinical isolates of Salmonella spp. analyzed so far.

36. Salmonella Function of SPI1
The function of SPI-1 is required for the invasion of nonphagocytic cells, an important virulence trait of S. enterica.
The SPI-1-encoded T3SS form needle-like surface appendages that can mediate the delivery of proteins by extracellular Salmonella organisms into host cells. A set of effector proteins encoded by SPI-1 and additional genes outside of SPI-1 are translocated.
SPI-1 expression responds to environmental stimuliand involves the SPI-1 encoded regulators HilC, HilD, HilA, and InvF

37. Salmonella Function of SPI2
SPI-1 and SPI-2 both encode T3SS for the translocation of virulence proteins into eukaryotic target cells, but the roles of SPI-1 and SPI-2 in virulence are entirely different.
SPI-2-encoded T3SS is required for the protection of the pathogen within the Salmonella-containing vesicle (SCV) against effector functions of the innate immunity. It help S. enterica to survive in phagocytic cells and to replicate within the Salmonella-containing vesicle in a variety of eukaryotic cells
It has been reported that SPI-2 function prevents the colocalization of the phagocyte oxidase as well as the inducible nitric oxide synthase with the SCV.

38. Salmonella interaction with host cells
Salmonella interaction with host cells. Salmonella enterica has evolved two virulence strategies using TTSS. The function of the SPI1-encoded TTSS is required for invasion of host cells and onset of diarrhoeal diseases. In contrast, the function of the SPI2-encoded TTSS appears to be restricted to intracellular Salmonella. Signals present in the phagosome induce SPI2 gene expression and the TTSS of SPI2 contributes to the intracellular survival and replication of Salmonella.

39. Salmonella SPI3 Structure:
SPI3 is inserted at the selC tRNA gene. The locus is about 17 kb, with an overall base composition similar to that of the core genome.
Two fragments of IS elements were located in the central region of SPI-3, and the GC content of genes within this locus is alternating.
SPI-3 shows heterogeneous structures in different subspecies of Salmonella spp.
Function:
The main virulence factor encoded by SPI-3 is the high-affinity magnesium transport system MgtCB, which is important for the intracellular phenotype of Salmonella to adapt to the microbicidal and nutrientpoor environment of the phagosome, which is limiting for purines, pyrimidines, particular amino acids, and Mg2+ .

40. SPI4 Salmonella Structure:
SPI4 forms an insertion of 25 kb in S. enterica serovar Typhimurium but is less complex in S. enterica serovar Typhi
Function:
Putative virulence factors in SPI-4 are encoded by genes with sequence similarity to genes encoding T1SS for toxins and a gene that is required for the survival in macrophages

41. SPI5 Salmonella Structure:
The locus is only 7 kb long, has a GC content of 43.6%, in contrast to 52% for the core genome of S. enterica, and is located adjacent to the serT tRNA gene.
Function:
encoding effector proteins for SPI-1 and SPI-2. SPI-5 harbors the gene for SopD, an effector protein of the SPI-1-encoded T3SS , as well as pipB, encoding an effector protein for the SPI-2-encoded T3SS.

42. Salmonella
Salmonella multi-drug resistant genomic island (SGI-1)
43 kb long and is flanked by DR. Further elements associated with DNA mobility, i.e., transposase, integrase, and excisionase genes with sequence similarities to transposon genes, have been detected.
SGI-1 can transfer between serovars in vitro through confugative mobilization and contributes to the spread of antibiotic resistance genes.

43. Helicobacter pylori pathogenicity island cag PAI (cytotoxin-associated gene)

44. H.Pylori Structure of cag PAI
the cag PAI had a size of 37 to 40 kb, flanked by direct repeats of 31 bp.
The locus has a GC content of 35%, in contrast to the 39% observed for the core genome
A gene for a tRNA has not been identified at the point of integration, but the glr gene (glutamate racemase) was disrupted by insertion of the PAI.

45. H.Pylori
In strains of intermediate virulence, various forms of deletions with the cag PAI were detected, and in certain strains the locus was separated into two portions, referred to as cagI and cagII

46. H.Pylori
Detailed analysis of the cagA loci in type I and type II strains indicated that the latter group showed deletions of a large chromosomal region.
There are no genes associated with DNA mobility within the cag PAI of type I strains. However, the presence of an IS 605 element within the cag PAI of strains with an intermediate virulence phenotype was observed.

47. H.Pylori Function of cag PAI
large portion of the cag PAI genes encode a functional T4SS and eight of them are homologues to components of the prototype T4SS.
the cag PAI encodes CagA, One of the defined H. pylori virulence factors.

48. Yersinia high-pathogenicity island (HPI)
Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI). The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin (耶尔森杆菌素） and can thus be regarded as an iron-uptake island.

49. High-pathogenicity island (HPI)
Yersinia
High-pathogenicity island (HPI)
it is a large chromosomal DNA fragment, varying in size from 36 kb in Y. pseudotuberculos isand to 43 kb in Y. enterocolitica
it carries genes essential for the expression of the high-pathogenicity phenotype, namely the yersiniabactin system involved in siderophore-mediated iron uptake;
it incorporates several repeated sequences (IS1328, IS1329, IS1222 and IS1400 or IS100) and a mobility gene (bacteriophage P4-like integrase gene)
it is bordered on one side by a tRNA locus (asn tRNA)
the G+C content of the open reading frames (ORFs) composing the yersiniabactin locus is close to 60%, and therefore much higher than the 46–50% reported for the remainder of the chromosome.

50. Yersinia 细菌 毒力岛 位置 大小 G+C% 特点 假结核耶氏菌 Yps HPI asn tRNA 35kb 59/47
与色素沉积片段连锁，末端具有DR17，一端有IS100
鼠疫耶氏菌
末端具有DR17，一端有IS100
小肠结肠炎耶氏菌
Yen HPI
45kb
57.5/47
末端具有DR17，无IS100，有IS328，329，400

51. Stap. aureus The SaPIs: mobile pathogenicity islands of Staphylococcus
Seven conserved PAI families in the S. aureus genome
designated vSa1(including SaPI1 and SaPI3); vSa2 (including SaPIbov); vSa3; vSa4 (including SaPI2); vSaa; vSab; and vSag.

52. Stap. aureus
Structure of SaPIs : a series of discrete 15–20 kb chromosomal elements that are mobilized at high frequencies
An example: The prototype of this family is the SaPI1 that was the first characterized SaPI. SaPI1 is 15.2 kb long, carries a tst gene, flanked by 17 bp DR sequences, and is inserted in an attc site close to the tyrB gene. The integration into the chromosome is facilitated by the presence of a functional integrase (int) gene encoded in the island. Remarkable features of SaPI1 are therefore its mobility and instability.

53. Stap. aureus SaPIs Function of SaPIs:
Staphylococcus PAI encoding TSST (中毒休克综合症毒素) (secreted virulence factors that function as superantigens toxins), The consequences of these TSST may include high fever, rash, vomiting, diarrhoea, renal and hepatic dysfunction and desquamation.
another important PAI is the Staphylococcus cassette chromosome mec (SCCmec), which encodes the methicillin resistance determinants MetI, MetR and MetA.

54. 尿路致病性大肠埃希菌（UPEC）PAI 特点： 大小为58-170kb； 携带毒力基因簇，能编码α-溶血素、P相关菌毛和Ⅰ型细胞坏死因子；
两侧具有同向重复序列和/或插入序列；
虽然各个毒力岛的细微结构不同，但都位于染色体的tRNA位点内；一些毒力岛上海发现有R质粒和P4噬菌体的序列；
G+C mol%均比宿主菌染色体低；
含有一些潜在的可移动成分如插入序列、整合酶基因等，可发生缺失。

55. UPEC PAI 菌株 PAI 位置 大小 G+C% 特点 UPEC 536 Pai I selC-tRNA 70kb 41/51
末端具有16bp的同向RS
Pai II
leuX-tRNA
90kb
末端具有18bp的同向RS
UPEC J96
Pai IV
pheV-tRNA
170kb
具有IS和R质粒，P4噬菌体序列，末端具有135bp的同向RS
Pai V
pheR-tRNA
106kb
具有IS和R质粒，P4噬菌体序列和OmpR类似结构
UPEC CF073
Pai VI
metV-tRNA
58kb
43/51

56. 肠致病性大肠埃希菌（EPEC） 肠出血性大肠埃希菌（EHEC）O157：H7
The LEE PAI
locus of enterocyte effacement

57. Structure of LEE:
35 kb
The LEE contains 41 ORFs and is organized as five polycistronic operons (LEE1–LEE5)
Analysis of the G + C content of the LEE (38%) showed that it is strikingly lower than that of the rest of the chromosome (50.8%).
The chromosomal integration site of LEE in the EPEC reference strain E2348/69 is the selC tRNA gene

58. Function of LEE PAI
Cause attaching-and-effacing (AE) lesions (粘附和脱落损伤）of the microvillus brush border of enterocytes
a T3SS, which is used as a molecular syringe to translocate effector proteins into host cells
the secreted translocator proteins (EspA, EspD and EspB) required for translocating effectors into host cells
the adhesin (intimin), which mediates intimate attachment to Tir on the host cell cytoplasmic membrane
the secreted effector proteins EspF, EspG,EspZ, EspH, Map and Tir, the intimin receptor, chaperonedby CesT

59. Vibrio cholerae pathogenicity island
VPI

60. Vibrio cholerae Structure of VPI
VPI is about 40 kb long and has a GC content of only 35% compared to an average GC content of the V. cholerae genome of 47 to 49%. Furthermore, VPI is inserted adjacent to ssrA, a gene with similarity to tRNA genes, and is flanked by att sites. Only one of these att sites remains after loss of VPI by naturally occurring deletions of the locus.

61. Vibrio cholerae Function of VPI
VPI encodes the toxin-coregulated pilus (TCP), a type IV pilus acting as an essential colonization factor by contributing to the adhesion of V. cholerae to the epithelial surface. In addition, TCP functions as the receptor for CTX, the bacteriophage harboring the cholera toxin genes. Thus, the presence of VPI is a prerequisite for the lysogenic conversion of nontoxigenic V. cholerae strains by CTX, an event that has contributed to the appearance of new toxigenic strains that caused cholera pandemics.
The VPI of V. cholerae is an excellent example of the contribution of bacteriophages to the distribution of PAI.

62. VPI encoded
CTX毒力单元，也可以质粒形式存在，亦可形成噬菌体分泌到菌体外，可在霍乱弧菌间水平转移，即以VPI毒力岛合成的Tcp菌毛为受体，感染无CTX毒力单元但携带VPI毒力岛的霍乱弧菌，并通过attRS位点整合到受体菌的染色体上，从而形成新的霍乱产毒株。