Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3943–3948, March 1998 Microbiology “Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli ANTHONY T. MAURELLI*†,REINALDO E. FERNA´NDEZ*, CRAIG A. BLOCH‡,CHRISTOPHER K. RODE‡, AND ALESSIO FASANO§ *Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, F. Edward He´bertSchool of Medicine, Bethesda, MD 20814-4799; ‡Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor, MI 48109-0656; and §Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201-1595 Edited by Herbert Tabor, National Institute of Diabetes, Bethesda, MD, and approved January 29, 1998 (received for review August 13, 1997) ABSTRACT Plasmids, bacteriophages, and pathogenicity formation of ‘‘black holes,’’ i.e., deletions of genes that are islands are genomic additions that contribute to the evolution detrimental to a pathogenic lifestyle. of bacterial pathogens. For example, Shigella spp., the caus- The four species of Shigella are so closely related to Esch- ative agents of bacillary dysentery, differ from the closely erichia coli that all of these bacteria could be considered related commensal Escherichia coli in the presence of a members of a single species. They share greater than 90% plasmid in Shigella that encodes virulence functions. However, homology by DNA–DNA reassociation analysis (2) and display pathogenic bacteria also may lack properties that are char- colinearity of their chromosomes such that gene transfer by acteristic of nonpathogens. Lysine decarboxylase (LDC) ac- conjugation and transduction and formation of recombinants tivity is present in '90% of E. coli strains but is uniformly between Shigella and E. coli occur with high efficiency (3). absent in Shigella strains. When the gene for LDC, cadA, was Nevertheless, Shigella spp. are frank pathogens that cause introduced into Shigella flexneri 2a, virulence became attenu- bacillary dysentery, whereas E. coli (with the exception of ated, and enterotoxin activity was inhibited greatly. The certain pathogenic clones) are commensals of the human enterotoxin inhibitor was identified as cadaverine, a product intestine. Of interest, one class of pathogenic E. coli, the of the reaction catalyzed by LDC. Comparison of the S. flexneri enteroinvasive E. coli (EIEC), resembles a genetic hybrid 2a and laboratory E. coli K-12 genomes in the region of cadA between E. coli and Shigella. EIEC carry plasmids with exten- revealed a large deletion in Shigella. Representative strains of sive homology to the virulence plasmid of Shigella and cause Shigella spp. and enteroinvasive E. coli displayed similar a diarrheal disease that is clinically similar to dysentery caused deletions of cadA. Our results suggest that, as Shigella spp. by Shigella (4). One of the striking biochemical features shared evolved from E. coli to become pathogens, they not only by EIEC and Shigella is a lack of lysine decarboxylase (LDC) activity. Whereas almost 90% of E. coli strains are LDC1 (5), acquired virulence genes on a plasmid but also shed genes via 2 deletions. The formation of these ‘‘black holes,’’ deletions of all strains of EIEC and Shigella spp. are LDC (6). This genes that are detrimental to a pathogenic lifestyle, provides observation suggested the possibility that absence of LDC an evolutionary pathway that enables a pathogen to enhance activity may be important for Shigella and EIEC virulence. virulence. Furthermore, the demonstration that cadaverine In this paper, we demonstrate that expression of LDC can inhibit enterotoxin activity may lead to more general activity by Shigella has no adverse effects on the invasive models about toxin activity or entry into cells and suggests an capability of this organism. However, cadaverine, produced by avenue for antitoxin therapy. Thus, understanding the role of the decarboxylation of lysine, acts as an inhibitor of Shigella black holes in pathogen evolution may yield clues to new enterotoxin activity. We further show that Shigella spp. and treatments of infectious diseases. EIEC have irreversibly lost LDC activity by genome deletion. These observations suggest that the creation of black holes (genome deletions) is a pathway that complements gene Virulence genes of bacterial pathogens may be encoded on acquisition in the evolution of bacterial pathogens. plasmids, bacteriophages, or the chromosome. Virulence is often multifactorial and coordinately regulated, and virulence genes tend to be clustered in the genome. Recently, the MATERIALS AND METHODS acquisition of pathogenicity islands was proposed as a major Bacterial Strains and Media. The strains used in this study mechanism in pathogen evolution (1). Pathogenicity islands are listed in Table 1. Strains were grown at 37°C in Luria– are regions on the genomes of certain pathogenic bacteria that Bertani medium (LB) with aeration, on LB agar, or on M9 are absent in nonpathogenic strains of the same or closely minimal salts with glucose (12). Media were supplemented related species and that contain large contiguous blocks of with thiamine (50 mgyml), spectinomycin (100 mgyml), kana- virulence genes. The addition of pathogenicity islands is being mycin (50 mgyml), or chloramphenicol (15 mgyml) as required. recognized as an important element in the evolution of To optimize enterotoxin production, bacteria were grown in bacterial pathogens through horizontal spread of virulence LB with ethylenediamine-N,N9-diacetic acid to chelate iron. genes similar to the horizontal transfer mediated by plasmids Supernatants from overnight cultures of bacteria were har- and bacteriophages. In this study, we report the existence of a complementary but inverse pathway that may enable com- This paper was submitted directly (Track II) to the Proceedings office. mensal bacteria to evolve toward a pathogenic lifestyle: the Abbreviations: EIEC, enteroinvasive E. coli; LDC, lysine decarboxyl- ase; LB, Luria–Bertani medium; PD, potential difference; Isc, short The publication costs of this article were defrayed in part by page charge circuit current. †To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Microbiology and Immunology, Uniformed Services University of accordance with 18 U.S.C. §1734 solely to indicate this fact. the Health Sciences, F. Edward He´bertSchool of Medicine, 4301 © 1998 by The National Academy of Sciences 0027-8424y98y953943-6$2.00y0 Jones Bridge Road, Bethesda, MD 20814-4799. e-mail: maurelli@ PNAS is available online at http:yywww.pnas.org. usuhsb.usuhs.mil. 3943 Downloaded by guest on September 25, 2021 3944 Microbiology: Maurelli et al. Proc. Natl. Acad. Sci. USA 95 (1998) y Table 1. Bacterial strains gassed with 95% O2 5% CO2. Once the tissue reached a m Source or steady-state condition, 300 l of sterile culture supernatant Strain Description reference was added to the mucosal side of the tissue. Sterile culture supernatant (300 ml) also was added to the serosal side to 2457T S. flexneri 2a wild type 7 preserve the osmotic balance. Supernatants of S. flexneri strain BS103 Plasmid-cured derivative of 2457T 8 2457T also were tested in the presence of either 300 mlof † BS529 2457T transformed with pCADA (cadA ) This study supernatant from S. flexneri strain BS529 or increasing con- BS573 BS103 zii-215::Tn10dCamRCP2 This study centrations of cadaverine. In a subset of experiments, the zjh-225::Tn10dSpcRCP2 intestinal epithelium was pretreated for 30 min with cadaverine MC4100 E. coli K-12 prototype 9 (300 mM), washed twice with fresh Ringer’s, and then exposed MG1655 E. coli K-12 prototype B. Bachmann* to 300 ml of 2457T supernatant. CAG18427 MG1655 zje-2241::Tn10 10 Once the tissues were exposed to the above treatments, the xM2115 MG1655 zii-215::Tn10dCamRCP2 11 x potential difference (PD; the difference in voltage between the M2125 MG1655 zjh-225::Tn10dSpcRCP2 11 mucosal and serosal sides of the tissue) was measured under xM2500 MG1655 zii-215::Tn10dCamRCP2 This study open-circuited conditions. The increase in voltage resulting zjh-225::Tn10dSpcRCP2 from the passage of 100 mA current was used to calculate the *E. coli genetic stock center. short circuit current (Isc; the amount of current needed to nullify the PD) and the tissue resistance from Ohm’s law (Isc 5 vested by centrifugation, filter-sterilized, and placed on ice PDytissue resistance) (20). until used. Genomic DNA Biophysical Techniques. Genomic DNA was pCADA is a plasmid that contains the wild-type cadA gene purified from 5.0-ml overnight cultures of E. coli::Tn10dRCP2 from E. coli K-12 under the transcriptional control of the lac and S. flexneri::Tn10dRCP2 mutants in a manner suitable for promoter (13). The cadA gene in pCADA is expressed con- yielding macrorestriction fragments (0.05–1.0 Mb) as de- stitutively in Shigella flexneri because of the absence of lac scribed (21). After digestion of agarose-embedded DNA with repressor in the organism and the plasmid vector. Cadaverine . I-SceI (Boehringer Mannheim) for 1 hr or with I-CeuI (Pan- ( 98% pure) was obtained from Sigma. Cultures of E. coli for vera, Madison, WI) overnight, according to the manufacturers’ measurement of LDC activity under inducing conditions were directions, reaction buffer was decanted, and dots were melted grown in medium buffered with 100 mM 4-morpholineethane- (70°C) and gently pipetted into sample wells in 1.3% agarose sulfonic acid to pH 5.5 (13). (Fastlane, FMC) gels for electrophoresis in a Bio-Rad DR-III Bacterial Genetics Techniques and Biochemical Assays. pulse field gel apparatus. Pulse times were ramped from 10 to Generalized transduction with P1 was as described (12). E. 13 s over 10 h and 60 to 65 s over 12 h at a field strength of 6 coli MG1655 mutants containing Tn10dSpcRCP2 or Vycm. After electrophoresis of samples, gels were analyzed as Tn10dCamRCP2 insertions were generated by electropora- described (22). tion with plasmids pGI300 or pGI310 as described (14). Genomic DNA Preparation and Southern Hybridization.