Virulence factors of septicemic Escherichia coli strains

Virulence factors of septicemic Escherichia coli strains

ARTICLE IN PRESS International Journal of Medical Microbiology 295 (2005) 455–462 www.elsevier.de/ijmm REVIEW Virulence factors of septicemic Esche...

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ARTICLE IN PRESS

International Journal of Medical Microbiology 295 (2005) 455–462 www.elsevier.de/ijmm

REVIEW

Virulence factors of septicemic Escherichia coli strains Daphna Mokady, Uri Gophna, Eliora Z. Ron Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel

Abstract Extraintestinal pathogenic Escherichia coli strains (ExPEC) are the cause of a diverse spectrum of invasive human and animal infections, often leading to septicemia. This review deals with the virulence genes of septicemic ExPEC strains. We discuss the meaning of a virulence gene and survey the genomic, genetic and physiological studies on these strains. Apparently, there are a few virulence factors, which are conserved in the septicemic strains, implying that they are essential for the infection. For the other virulence-related genes a high level of diversity is observed, demonstrating that all stages of the infection can be mediated by a number of alternative virulence factors. The variable profile of virulence genes in septicemic E. coli strains, as well as a prevalence of mobility-related sequences point out the existence of a ‘‘mix and match’’ combinatorial system. r 2005 Elsevier GmbH. All rights reserved.

Contents Defining a virulence factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Septicemic Escherichia coli strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agenomic approach for identifying virulence factors of septicemic strains . Distribution of the specific sequences among the septicemic strains . . . . . . Bona fide virulence factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ColV plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iron acquisition systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adhesins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretion systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Curli – a virulence factor? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Defining a virulence factor Virulence factors involve mechanisms, which enable pathogenic bacteria to cause disease. However, defining which factors constitute virulence factors is by no means Corresponding author. Tel.: +972 3640 9379; fax: +972 3641 4138.

E-mail address: [email protected] (E.Z. Ron). 1438-4221/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2005.07.007

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an easy task. One way of identifying virulence factors involves mutagenesis of the pathogens and determining which of the mutations resulted in reduced virulence. Experiments of this type led to a large number of publications indicating that the heat shock response, e.g., is essential for virulence, as mutants in various components of the heat shock response are clearly less virulent (Gophna and Ron, 2003). Consider the example

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of the transcriptional activator of the heat shock genes – s32 – responsible for expression of heat shock genes at elevated temperatures. Mutants in s32, as well as in genes coding for heat shock proteins, have reduced virulence. Yet, it is known that deletion mutants in the rpoH gene, coding for s32 grow very poorly at temperatures above 22 1C. As experiments to determine the degree of virulence are carried out in vivo and involve injection into animals with a body temperature of 37 or 42 1C for mammals and birds, respectively, what do the results mean? They probably constitute another set of data indicating that mutants in the heat shock response do not grow well above 22 1C, and bear no indication whatsoever to the level of virulence of these mutants. Therefore, although components of the heat shock response have a profound effect on virulence they should not be classified as a virulence factor because they also affect other pathways, which are not associated with virulence, and therefore are not ‘‘virulence specific’’. However, such a restrictive approach will also exclude many ‘‘classical’’ virulence factors such as adhesins, which besides mediating adherence of bacteria to host tissues also facilitate binding to solid surfaces, and flagella which contribute to virulence but are also very important for fitness of free-living bacteria. One should also keep in mind that the relative importance of a virulence factor can only be evaluated in context of other elements utilized by the bacterium in the infection process. Thus, the presence of a single virulence factor rarely makes a strain virulent, and only a combination of virulence factors will determine its ability to cause disease. Genomics offers an interesting tool for defining virulence factors – they can be defined as specific factors which contribute to virulence and are encoded by genes which are present in the pathogen and absent from nonpathogenic strains. Yet, as we will show in this review, in many cases the determining factor is not the presence or absence of a virulence-related gene, but its level of expression which can vary between the pathogenic and non-pathogenic strains.

Septicemic Escherichia coli strains Pathogenic E. coli strains cause intestinal or extraintestinal infections in many host species. Strains that cause extraintestinal infections are involved in a diverse spectrum of diseases, including urinary tract infections (UTI), newborn meningitis (NBM) and septicemia (Babai et al., 1997; Hacker et al., 1997; Meier et al., 1996; Ron et al., 1990; Tschape and Hacker, 1991; Zingler et al., 1993). Septicemia in humans is usually secondary to UTI or respiratory diseases. It is also an important disease of farm animals, where colisepticemia

in calves and lambs and especially avian colisepticemia constitute a disease with significant economic importance. Avian pathogenic E. coli (APEC) strains bring about serious extraintestinal disease of poultry causing high mortality and morbidity in chickens and turkeys – leading to considerable economic losses (Ron et al., 1990; Yerushalmi et al., 1990). These severe infections are caused when colonization of the upper respiratory tract and airsacs is followed by spreading of bacteria to internal organs (pericarditis, perihepatitis) and by entry into the bloodstream – septicemia – which is often fatal (Dho-Moulin and Fairbrother, 1999). APEC strains, which usually belong to serotypes O78, O1 and O2, have been known to harbor various virulence factors, most of which are also characteristic of human extraintestinal strains. E. coli is a leading cause of bloodstream infections in nursing homes (Mylotte et al., 2002), hospitalized persons (Siegman-Igra et al., 2002) and children, especially newborns (Kim et al., 2002). Avian colisepticemia therefore provides a unique advantage by enabling identification and characterization of factors important for pathogenesis of sepsis of both mammals and birds. The roles of these factors in virulence can later be tested in vivo in a natural infection model.

Agenomic approach for identifying virulence factors of septicemic strains The best procedure for identifying unique genes is by whole genome sequencing. However, as no whole genome was sequenced for septicemic E. coli strains, the molecular procedure of suppression subtractive hybridization (SSH) was used as a faster genomic approach. This procedure allows the comparison of two genomes, and detection of specific sequences that are present exclusively in one of them. This technique has been shown to be extremely efficient in detecting genomic sequences and genomic islands that are strain specific and was used to identify virulence factors by comparing pathogenic and non-pathogenic bacterial strains of the same species (Walker and Verma, 2002; Akopyants et al., 1998; Janke et al., 2001; Zhang et al., 2000). The genome of the non-pathogenic laboratory strain K-12 was subtracted from the genome of two septicemic strains belonging to serotypes O2 and O78, and unique sequences were studied. Analysis was performed on 158 sequences obtained by SSH and verified by PCR. Table 1 presents the functional distribution of the unique sequences. The unique sequences include a high ratio of virulence-related genes, or genes with no blast homology, which could also be virulence related. The presence

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Table 1. Distribution of unique sequences in SSH libraries of septicemic strains Function

%

Known virulence genes Putative virulence genes Mobility-related genes Unkown function

9 16 29 34

of numerous mobility-associated sequences, such as transposases and integrases, along with phage-related sequences, plasmid sequences and insertion sequencesassociated sequences indicates the presence of genomic areas that were acquired horizontally, potentially related to virulence. The prevalence of sequences associated with genomic plasticity in the subtractive libraries supports the assumption that the pathogenic strains evolved by processes involving genome remodeling and horizontal acquisition of genomic regions from other pathogenic bacteria.

Distribution of the specific sequences among the septicemic strains Fig. 1 shows the distribution of 45 specific sequences among 14 septicemic isolates of strains O2 and O78 (Mokady et al., 2005). The results clearly indicate that the sequences are patchily distributed, most of them only present in a small subset of strains, and may therefore have been acquired by fairly recent horizontal gene transfer. A few genes probably represent an ancient transfer event, as they are found in all the pathogenic strains tested, but not in K-12. The fact that not many virulence factors are common in all the septicemic strains examined is unexpected, since there is wide gene distribution even between strains that cause the same disease (Mokady et al., 2005). The findings imply that the various strains are using different factors with similar roles in the various stages of the infection process. Thus, each step in the infection process can be mediated by a number of alternative virulence factors, and each strain may have a unique combination of such factors. This assortment of virulence genes is apparently made possible by the variety of genetic factors contributing to genome plasticity, such as plasmids, phages and transposable elements. The fact that extant E. coli strains vary so much in the content of their genomes, indicates that this ‘‘mix and match’’ combinatorial approach has been a successful evolutionary strategy for these species, which can colonize many different tissues and hosts.

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Previous genomic comparisons of E. coli strains have shown great differences in gene content. However, none of the comparative genomics of sequenced E. coli strains compared strains, which cause the same disease, and target the same host tissues. The only exception is in the two genome sequences of O157:H7. In these strains, the virulence factors and genes coding for them show a very high degree of similarity (Jin et al., 2002; Perna et al., 2001). These results are in contrast to our findings in the septicemic O2 and O78 serogroups, showing a very high level of genome plasticity. An additional and important conclusion from these data is the indication that the large pool of variable virulence genes is accessible to septicemic bacteria, independent of the host. It therefore remains to be determined whether there is host specificity among the septicemic extraintestinal pathogenic E. coli (ExPEC) strains, and which factors control the zoonotic risk.

Bona fide virulence factors The comparison of septicemic strains enabled the identification of a few shared traits, which are present in all the strains, in one form or another. These common virulence factors are presumably important, or even essential, for the infection process. The data presented in Table 2 compare the virulence factors that are thought to participate in the different infection stages of isolates of strains O2 and O78. The two groups of strains have adherence pili, but of different types – O2 codes for P pili (Mokady et al., 2005) while O78 codes for AC/I pili (Babai et al., 2000), for a non-fimbrial putative adhesin (Mokady et al., 2005) and for long polar fimbriae (Lpf) (Ideses et al., 2005). Another virulence factor that varies between the strains is the capsule. While O2 strains possess a K-1 polysaccharide capsule, O78 strains lack such capsule, but are enveloped by a type IV capsule (KrallmannWenzel and Schmidt, 1994; Mangia et al., 1999; Ron et al., unpublished results). All the septicemic strains carry a plasmid encoding Colicin V (ColV plasmid), known to encode also the aerobactin iron uptake system as well as serum resistance (Valvano and Crosa, 1984; Waters and Crosa, 1986, 1991; Zgur-Bertok et al., 1990). Other common virulence factors are additional iron uptake systems, chromosomally located, and genes of the type III secretion system, the function of which in translocating virulence-related proteins into the host has so far not been proven (Ideses et al., unpublished). It should be noted that although there were a few reports on potential toxins (Parreira and Gyles, 2002, 2003; Salvadori et al., 2001), extracellular toxins do not appear to be a critical virulence factor in the septicemic strains examined so far.

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120

% of O78 strains with sequence

100

80

60

40

20

0 120

% of O2 strains with sequence

100

80

60

40

20

0 Sequence

Fig. 1. Distribution of specific sequences in O2 and O78 ExPEC strains. The distribution of specific sequences was determined by PCR in 14 septicemic E. coli strains. The sequences used were identified by SSH and found only in pathogenic strains. These sequences were also found by BLAST to belong to genes associated with virulence or genes of unknown function.

ColV plasmids Most septicemic strains contain ColV plasmids which carry genes encoding the aerobactin iron uptake system as well as genes coding for serum resistance (Valvano and Crosa, 1984; Waters and Crosa, 1986, 1991; ZgurBertok et al., 1990). A recent study of a ColV plasmid

isolated from an avian septicemic strain (Gophna et al., 2003) indicated that this plasmid – pO78V – is chimeric, containing genes of ColV, which belongs to the IncF incompatibility group together with several genes typical to IncI plasmids. This plasmid, which is conjugative and carries tetracycline resistance, also encodes type IV pili, already shown to be important for adhesion to and

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Table 2.

Virulence factors present in septicemic strains

Function

Virulence factor/gene

Demonstrated in strains of serotype

Iron uptake

Aerobactin Yersiniabactin IroN receptor SitABCD

O2, O78 O2, O78 O2, O78 O2

Serum resistance

ColV plasmid

O2, O78

Adhesins

Type I pili AC/I pili P pili Non-fimbrial adhesin Long polar fimbriae Curli Type IV

O2, O78 O78 O2, O78 O78 O78 O2, O78 O78

Capsule

K-1 Type IV

O2 O78

invasion of epithelial cells in Salmonella typhimurium serovar Typhi (Kim and Komano, 1997). These additional genes broaden the spectrum of virulence factors beyond the previously known virulence properties of ColV plasmid (Gophna et al., 2003).

Iron acquisition systems Iron is an essential cofactor for bacterial metabolism. The fact that iron in body fluids (e.g., plasma) is bound to host proteins such as transferrin, reduces its availability to far below concentrations allowing bacterial survival. Septicemic E. coli strains cope with the scarcity of iron by producing low-molecular-weight compounds (siderophores) which bind iron with high affinity and compete efficiently with host proteins. Once iron-bound, these chelators are taken up into the bacteria by specific receptors on their membranes. Aerobactin production by APEC strains was correlated with virulence (Lafont et al., 1987) and several reports indicated that most virulent strains carried this iron uptake system (Dozois et al., 1992; Gophna et al., 2001c; Janben et al., 2001). The aerobactin cluster is often located on ColV plasmids (Valvano, 1992), which can confer other virulence properties such as serum resistance or type IV pili expression (Gophna et al., 2003). Recent reports demonstrate the existence of another iron uptake system – similar to that located on the high pathogenicity island (HPI) typical of pathogenic Yersinia. The HPI-encoded iron uptake system of septicemic strains includes genes for the biosynthesis of the siderophore yersiniabactin and genes coding for its receptor. The presence of the yersiniabactin iron uptake system was shown in many

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ExPEC strains (Karch et al., 1999; Schubert et al., 1998) and in the majority of avian pathogenic strains (Gophna et al., 2001c, 2003; Janben et al., 2001). It has been shown that yersiniabactin production contributes to virulence in mice of two extraintestinal E. coli strains (Schubert et al., 2002). Most of the septicemic strains contain both iron uptake systems, aerobactin and yersiniabactin. In addition, many septicemic strains contain an additional receptor – IroN – which binds the enterobactin siderophore with higher affinity (Russo et al., 1999, 2002). These findings suggest that multiple siderophores may prove beneficial under different conditions. Thus, one siderophore could have higher affinity or stability than the other in certain tissues of the host. Since yersiniabactin has higher affinity for iron than aerobactin, it may prove beneficial for the bacterium under particularly severe iron depletion. Alternatively, either siderophore may have some immuno-modulatory role besides its activity as iron-scavenger (Schubert et al., 2002).

Adhesins Colonization of the host tissues is mediated by specific adherence. This attachment is mediated by adhesins located on fimbriae (or pili) or by non-fimbrial adhesins. It is assumed that this stage determines the host and tissue specificity and increases the binding efficiency. Septicemic strains contain a variety of adherence factors. Human NBM O78 strains carry the P pili (Ron et al., 1990), septicemic strains from calves and lambs carry the K-99 pili, while avian strains often carry the avian specific adherence fimbriae – AC/I (Naveh et al., 1984; Ron et al., 1990; Yerushalmi et al., 1990). The latter are related to S-fimbriae from human NBM and sepsis, which bind sialylgalactosides (Babai et al., 1997). However, the minor subunits of the two kinds of pili differ in primary structure. In addition, avian septicemic strains of serotype O78 often carry a non-fimbrial adhesin and the Lpf (Ideses et al., 2005; Baumler et al., 1996a, b).

Secretion systems Many septicemic strains contain genes of the type III secretion system, which mediates the translocation of virulence-related bacterial proteins into host cells (China and Goffaux, 1999; Kenny, 2002; Nougayrede et al., 2003). Yet, it has not yet been shown that this secretion system is functional or required in septicemic strains (Ren et al., 2004; Ideses et al., unpublished). An interesting observation is the secretion of proteins, including outer membrane proteins (Gophna et al., 2004). The secretion of OmpA by a septicemic E. coli

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O78 strain may play a role in virulence, as OmpA has been shown to bind neutrophil elastase, which is bactericidal.

Curli – a virulence factor? Curli fibers are thin aggregative surface fibers which were recently found to be amyloid (Chapman et al., 2002). These fibers are expressed by many pathogenic isolates of E. coli, including avian isolates, as well as laboratory strains (Gophna et al., 2001b; Olsen et al., 1989; Uhlich et al., 2001, 2002). Curli fibers are adhesive and bind several host molecules including laminin (Olsen et al., 1993), fibronectin (Olsen et al., 1989), plasminogen (Sjobring et al., 1994), human contact phase proteins (Ben Nasr et al., 1996) and major histocompatibility complex class I molecules (Olsen et al., 1998). Curli-negative mutants showed reduced adherence to chick explant tissues (La Ragione et al., 2000b) as well as lower colonization, invasion and persistence in 1-day-old chicks (La Ragione et al., 2000a), compared to their wild-type isogens. It has been demonstrated that curli fibers play a role in invasion of eukaryotic cells by E. coli probably via their binding to fibronectin (Gophna et al., 2001a, b, 2002). All these factors suggest that curli is a virulence factor. However, curli are also coded for by non-virulent strains, including E. coli K-12. Moreover, sequence analysis showed that the cluster responsible for internalization in the virulent strain is nearly identical to the csg gene cluster encoding K-12 curli fiber formation. This finding raises a few questions, since this cluster is carried by most E. coli strains including many nonpathogenic isolates. One possible answer is the finding that most known E. coli strains do not express curli under host-like conditions – i.e., high to moderate osmolarity and high temperature, while the septicemic strain does express curli under such conditions (Gophna et al., 2001b). The case of curli indicates that the definition of virulence genes should be broadened. It should include genes that contribute to virulence and are present only in pathogenic strains as well as genes which are expressed only in the pathogenic strains under host conditions.

Acknowledgements This work was supported by the Manja and Morris Leigh Chair for Biophysics and Biotechnology, the Israel Center for Emerging Diseases and the European Community project COLIRISK.

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