The cell division genes (ftsE and X) of Aeromonas hydrophila andtheir relationship with opsonophagocytosis

Susana Merino, Mar|a Altarriba, Rosalina Gav|n, Luis Izquierdo, Juan M. Tomas *Departamento Microbiolog|a, Facultad Biolog|a, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain

Received 18 January 2001; received in revised form 12 March 2001; accepted 19 March 2001

Abstract

A transposon mutant from Aeromonas hydrophila AH-3 was obtained which was highly resistant to opsonophagocytosis. The mutationwas identified in the ftsE gene and we characterised the operon ftsY, E and X from this bacterium. These genes, as in enteric bacteria, areneighbours to rpoH. The A. hydrophila ftsE and X genes were fully able to complement Escherichia coli ftsE mutants, and also complementthe opsonophagocytosis-resistant phenotype of the A. hydrophila mutant strain. This phenotype seems to be related to the filamentousphenotype at 37³C exhibited by the A. hydrophila ftsE mutant. ß 2001 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Opsonophagocytosis resistance; ftsE ; Cell morphology; Aeromonas hydrophila

1. Introduction

Mesophilic motile Aeromonas species are opportunisticand primary pathogens of a variety of aquatic and terres-trial animals, including humans; the clinical manifesta-tions range from gastroenteritis to soft-tissue infections,including septicemia and meningitis [1]. Serogroup O:34strains of mesophilic Aeromonas spp. have been recoveredfrom moribund ¢sh [2] and from clinical specimens [3] ;O:34 is the most common Aeromonas serogroup [4], ac-counting for 26.4% of all infections. Previous investiga-tions have documented O:34 strains as important causesof infections in humans [4,5]. The varied clinical picture ofAeromonas infections, and gastroenteritic illness in partic-ular, suggests that complex pathogenic mechanisms occurin aeromonads.

Phagocytes are host cells that are adapted speci¢cally toengulf and destroy bacteria or other foreign particulatematter. Two types of professional phagocytes are de-scended from bone marrow stem cells : polymorphonuclearleukocytes (PMNLs) and macrophages. Opsonisationwithout speci¢c antibody takes place following depositionof complement opsonin C3b on the bacterial surface, and

the attachment to the C3b receptor on the phagocytewhich is known as opsonophagocytosis. Opsonophagocy-tosis is the most e¤cient mechanism to eliminate foreignbacteria.

In this study we isolate a transposon mutant from Aero-monas hydrophila AH-3 (serogroup O:34) [2] highly resis-tant to opsonophagocytosis, which allowed us to charac-terise the gene/s involved in this resistance that have beenpreviously related to cell morphology (¢lamentous pheno-type).

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

Bacterial strains and plasmids used in this study arelisted in Table 1. Escherichia coli strains were grown onLB Miller broth and LB Miller agar, while Aeromonasstrains were grown on tryptic soy broth or agar [6]. Am-picillin (Ap; 50 Wg ml31), chloramphenicol (Cm; 25 Wgml31), kanamycin (Km; 25 Wg ml31), rifampicin (Rif ;100 Wg ml31) or tetracycline (Tc; 20 Wg ml31) were addedto the di¡erent media when needed. Mini-Tn5 mutantsfrom Rif-resistant A. hydrophila (wild-type strains) wereobtained by conjugation with E. coli strain S17-Vpir carry-ing plasmid pUTmini-Tn5Km1 selecting for Rif and Kmresistance [7]. Plasmid pACYC-FTS was obtained from

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 4 4 - 6

* Corresponding author. Tel. : +34 (93) 4021486;Fax: +34 (93) 4110592; E-mail : juant@porthos.bio.ub.es

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www.fems-microbiology.org

COS-FTS after polymerase chain reaction using the fol-lowing oligonucleotides (5P-AAGTTCCAGATCCCGA-TCC-3P and 5P-GATCCGTGTCTCAAAATCC-3P), whichproduced a DNA product of 1953 bp. The DNA band wasblunt-ended and ligated to the EcoRV site of pACYC184selecting for Cm resistance. Plasmid pACYC-FTS con-tained only the complete ftsE and X-like genes from A.hydrophila AH-3.

2.2. Opsonophagocytosis

Opsonophagocytosis in the absence of speci¢c antiserumwas performed as previously described [8,9]. Brie£y, 0.2 mlof bacterial suspension (ca. 107 cfu ml31) was mixed with0.2 ml of human PMNLs (ca. 106 cells ml31) and 0.05 mlof non-immune human serum in Hanks' balanced salt so-lution, and incubated at 37³C for 30 min. Usually humanPMNLs were obtained from human blood freshly ob-tained [8]. In some cases, human PMNLs were substitutedby mouse bone marrow-derived macrophages obtainedfrom the cell line L929 (mouse ¢broblast) by growing onL-cell-conditioned medium as previously described [10].Bacterial counts (% survival) were done in some cases byserial dilution and colony enumeration on agar plates. Inother cases, prior to centrifugation of the eukaryotic cells(400Ug, 5 min at 4³C), 10 ml of cold Hanks' balanced saltsolution were added to the mixture, and the resuspendedpelleted cells in the same salt solution were smeared ontoglass slides and stained with LeukoStat stain. One hun-dred human PMNLs on each of duplicate slides were ex-amined to determine the percentage phagocytosis (%P)and the phagocytosis index (PI) as previously described[8,9].

2.3. C3 binding

The interaction between whole A. hydrophila cells andcomplement C3b was quanti¢ed using an enzyme immu-noassay as previously described [11].

2.4. General DNA methods

DNA manipulations were carried out essentially as pre-viously described [12]. DNA restriction endonucleases, T4DNA ligase, E. coli DNA polymerase (Klenow fragment),and alkaline phosphatase were used as recommended bythe suppliers.

2.5. Construction of an A. hydrophila AH-3 genomic library

A. hydrophila AH-3 genomic DNA was isolated andpartially digested with Sau3A as described by Sambrooket al. [12]. Cosmid pLA2917 [13] was digested with BglII,dephosphorylated and ligated to Sau3A genomic DNAfragments. DNA packaging using Gigapack Gold III(Stratagene) and infection of E. coli DH5K were carried

out as previously described. Recombinant clones were se-lected on LB agar plates supplemented with Tc.

2.6. Southern blot and colony blot hybridisations

Southern blotting was performed by capillary transfer[12]. For colony blot hybridisations, the colonies weretransferred onto Hybond N� (Amersham) nylon mem-brane and lysed according to the supplier. Probe-labelling,hybridisation, and detection were carried out using theenhanced chemiluminescence-labelling and detection sys-tem (Amersham) according to the manufacturer's instruc-tions.

2.7. DNA sequencing

Primers used for DNA sequencing were purchased fromPharmacia LKB Biotechnology. Double-stranded DNAsequencing was performed, using the Sanger dideoxy-chaintermination method [14] with the Abi Prism dye termina-tor cycle sequencing kit (Perkin Elmer).

2.8. DNA and protein sequence analysis

The DNA sequence was translated in all six frames, andall open reading frames (ORFs) greater than 100 bp wereinspected. Deduced amino acid sequences were comparedwith those of DNA translated in all six frames from thenon-redundant GenBank and EMBL databases using theBLAST network service at the National Center for Bio-technology Information [15]. Multiple sequence align-ments and determination of putative terminator sequenceswere done, using the PileUp and Terminator programsfrom the Genetics Computer Group package (Madison,WI, USA) in a VAX 4300.

2.9. Nucleotide sequence accession numbers

The nucleotide sequences of the genes described herehave been assigned the following GenBank accession num-ber: AF334761.

3. Results

After mutagenesis of a Rif-resistant isolate (AH-405) ofthe A. hydrophila wild-type strain (AH-3, serogroup O:34,we selected mutants (Kmr) resistant to opsonophagocyto-sis using non-immune human serum for opsonisation andmacrophages derived from the cell line L929. Mutant AH-414 was selected because of its resistance to opsonopha-gocytosis in a standard assay (Table 2) among 1200 mu-tants initially screened. As shown in Table 2, mutant AH-414 showed an 85% decrease in the PI and a 80% decreasein the %P in comparison to the wild-type strain. The de-crease in both indices is linked to a very large increase in

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cell survival measured by the plate dilution count. How-ever, the amount of C3b deposited on whole cells afterincubation for 30 min at 37³C with non-immune humanserum was similar in the wild-type and the mutant strain(ca. 0.45 arbitrary units (OD405 enzyme immunosorbentassay measurements)).

Southern blot analysis using a speci¢c probe for thetransposon demonstrated that mutant AH-414 had a sin-gle copy of the mini-transposon in its genome. Cloning ofthe mini-transposon-containing fragment from the ge-nomic DNA of the mutant (EcoRV digestion) in pBlue-script (Stratagene) and sequencing of the mini-transposon-£anking regions showed ca. 60% of identity and over 70%similarity with ftsE genes (cell division ATP-binding pro-tein) of E. coli, Vibrio cholerae, Pseudomonas aeruginosa,Haemophilus in£uenzae and others.

Using the following oligonucleotides from the mini-Tn5Km1, 5P-AGATCTGATCAAGAGACAG-3P and 5P-ACTTGTGTATAAGAGTCAG-3P, and the universal oli-

gonucleotides from the plasmid vector T3 and T7, wesequenced the complete 4-kb EcoRV fragment cloned inorder to design a DNA probe for this gene (ftsE-like genefrom A. hydrophila AH-3).

3.1. Cloning of an A. hydrophila AH-3 genomic regionencoding for ftsE in E. coli K12 strains

A. hydrophila AH-3 is a typical strain of serogroup O:34[2]. A cosmid-based genomic library of A. hydrophila AH-3 was constructed and introduced into E. coli DH5K asindicated in Section 2. Tc-resistant (20 Wg ml31) cloneswere screened by colony blotting using a DNA probe pre-viously obtained from the region close to the mini-Tn5location in mutant strain AH-414. We found several re-combinant positive clones, of which clone COS-FTS wasrepresentative, able to complement the AH-414 mutation(Table 2).

Table 1Bacterial strains, cosmids and plasmids used

Strain, cosmid or plasmid Relevant characteristics Source or Reference

E. coliDH5K F-endA hsdR17 (rk3 mk+) supE44 thi-1 recA1 gyr-A96 x80lacZ [23]XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (FP proAB lacIZ M15 Tn10) (Stratagene)S17-1 hsdR pro recA, RP4-2 in chromosome Km: :Tn7 (Tc: :Mu) [24]RG60 MG1655 ftsE: :kan recA : :Tn10 [18]JS10 ftsE2000, cold-sensitive (B. Bachmann)

Aeromonas sp.AH-3 A. hydrophila wild-type, serogroup O:34 [2]AH-405 Rif-resistant mutant derived from AH-3 [19]AH-414 AH-405 ftsE mini-Tn5 mutant (this work)AH-418 Mutant AH-414 complemented by pACYC-FTS plasmid (this work)

PlasmidspUTmini-Tn5Km1 Kmr [7]pLA2917 Tcr, Kmr [13]COS-FTS pLA2917 with 20-kb chromosomal AH-3 Sau3A insert (ftsE and X-like genes) (this work)pBluescript Apr (Stratagene)pACYC184 Tcr, Cmr [12]pACYC-FTS pACYC184 with 1.953 bp of AH-3 (ftstE and X-like genes) (this work)

r, resistant

Table 2Opsonophagocytosis of di¡erent A. hydrophila strains opsonised with 90% non-immune human serum for 30 min at 37³C

Strain PIa %Pb % Survivalc

AH-3 (wild-type) 375 þ 26 78 þ 6 6 0.1AH-405 (AH-3 Rifr) 369 þ 28 76 þ 7 6 0.1AH-414 (ftsE) 42 þ 8 15 þ 3 37 þ 5AH-414+COS-FTS 345 þ 23 72 þ 8 6 0.1AH-418 (AH-414+PACYC-FTS) 358 þ 22 75 þ 6 6 0.1AH-414+pACYC184 40 þ 6 13 þ 4 34 þ 4

All results are the average of three independent experiments þ standard deviations.a PI = phagocytic index, the number of bacteria associated with 100 PMNLs [8,9].b %P = percentage phagocytosis, (the number of PMNLs containing bacteria/total number PMNL)U100 [8,9].c Percentage survival of viable bacteria as described in Section 2.

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3.2. ftsY, E, X and rpoH DNA sequence

Using internal primers designed from the ftsE gene wecompleted the DNA sequence of a 5.8-kb fragment fromthe cosmid COS-FTS. Four complete ORFs transcribed inthe same direction were obtained as shown in Fig. 1.

For ORF1 (FtsY homologue) the deduced amino acidsequence was found to be highly identical/similar (%) tothe cell division membrane protein FtsY of E. coli (73/85),V. cholerae (73/82) gene VC0147 [16], H. in£uenzae (67/80), P. aeruginosa (62/75) and others. This protein (FtsY)is one of the ¢lamentous temperature-sensitive (fts) pro-teins, which is a signal recognition particle-type of GTPaseinvolved in protein targeting.

For ORF2 (FtsE homologue) the deduced amino acidsequence showed the following identities and similarities(%) to the cell division ATP-binding protein of: E. coli(67/81), V. cholerae (66/80) gene VC0148, P. aeruginosa(58/74), H. in£uenzae (58/72) and others. The mini-Tn5transposon of mutant strain AH-414 was located on thisgene (Fig. 1). FtsE is the ATP-binding protein of a com-plex that bears characteristics of an ATP-binding cassette(ABC)-type system (¢rst component).

For ORF3 (FtsX homologue) the deduced amino acid

sequence was found to be highly identical/similar (%) to acell division membrane protein FtsX of P. aeruginosa (37/54), V. cholerae (36^53) gene VC0149, H. in£uenzae (31/54,E. coli (31/50) and others. FtsX is the integral membraneprotein of a complex that bears characteristics of an ABC-type system (second component), which clearly explainedthe reduced identity/similarity in comparison with the oth-er ORFs.

ORF4 (RpoH homologue) had a deduced amino acidsequence which showed the following identities and simi-larities (%) to the RpoH protein of: Proteus mirabilis (72/85), Serratia marcescens (72/84), V. cholerae (70/85) geneVC0150, E. coli (69/83) and others. RpoH is a RNA poly-merase sigma-32 (heat shock regulatory protein).

3.3. The ftsEX genes from A. hydrophila AH-3:complementation assays

In order to study the functionality of the ftsEX genes ofA. hydrophila, we used two ftsE mutants from E. coli JS10[17] and RG60 [18] whose ¢lamentous phenotype temper-ature conditions are shown in Table 3. E. coli JS10 andRG60 mutant strains carry a pACYC-FTS plasmid (pA-CYC184 carrying the ftsEX genes from A. hydrophila AH-3, see the plasmid construction in Section 2) recovered thesame phenotype as the wild-type E. coli (non-¢lamentousat any temperature assayed, Table 3). The same mutantstrains carrying the plasmid vector alone (pACYC184)showed no phenotypic changes. An example of the ¢la-mentous phenotype observed and the complementationstudies is shown in Fig. 2. Furthermore, strain RG60 car-rying the pACYC-FTS plasmid was able to grow in theabsence of NaCl at 42³C, while as previously shown nei-ther strain RG60 nor RG60 carrying pACYC184 plasmidwere able to do this [18].

A. hydrophila mini-Tn5 mutant (AH-414) carrying the

Fig. 1. Genetic organisation of the A. hydrophila AH-3 cloned and se-quenced DNA fragment from COS-FTS. Predicted ORFs were namedafter their homologues in other bacterial species. The arrowhead indi-cates the site of mini-Tn5Km1 insertion in the mutant chromosome.Horizontal arrows indicate the direction of transcription. Some of therestriction sites are also shown.

Table 3Phenotypes of di¡erent strains cultured at several growth temperatures

Strain Growth temperature

20³C 30³C 37³C 42³C

A. hydrophilaAH-3 (wild-type) NF NF NF NTAH-405 (AH-3 Rifr) NF NF NF NTAH-414 (ftsE) NF NF F NTAH-414+COS-FTS NF NF NF NTAH-418 (AH-414+PACYC-FTS) NF NF NF NTAH-414+pACYC184 NF NF F NT

E. coliDH5K (wild-type) NF NF NF NFJS10 (ftsE) F F NF NFJS10+pACYC-FTS NF NF NF NFJS10+pACYC184 F F NF NFRG60 (ftsE) NF NF F FRG60+pACYC-FTS NF NF NF NFRG60+pACYC184 NF NF F F

F = ¢lamentous phenotype; NF = non ¢lamentous phenotype; NT = not tested.

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pACYC-FTS plasmid was obtained after a triparentalmating using E. coli DH5K plus pACYC-FTS plasmid asa donor, E. coli DH5K plus pRK2073 (helper) plasmid [19]and the mutant as recipient strain selecting for Cm andKm resistance. The complemented strain (AH-418) was assensitive to opsonophagocytosis as the wild-type strain,while the mutant strain carrying the plasmid vector alone(pACYC184) showed a similar resistance to opsonophago-cytosis to the mutant strain AH-414. The molecular genet-ic analysis performed previously prompted us to study themorphology of the mutant strain AH-414 at severalgrowth temperatures. The mutant (AH-414) showed a ¢l-amentous phenotype at 37³C but not at 30³C (Fig. 2), andthe viability of this mutant strain at high temperature wasindependent of the NaCl concentration. Identical pheno-types were observed when AH-414 carried the plasmidvector pACYC184, but the complemented strain AH-418showed the same phenotype (not ¢lamentous at any tem-perature) as the AH-3 wild-type strain.

4. Discussion

In this study we used an opsonophagocytosis assay forinitial identi¢cation of genes involved in opsonophagocy-tosis resistance compared with a sensitive wild-type strain(AH-3; serogroup O:34). This strain is resistant to com-plement lysis, but is clearly opsonised by C3b via the clas-sical complement pathway [11]. For this reason it is op-sonophagocyted by human PMNLs. The isolation of amini-transposon mutant with a high degree of opsonopha-gocytosis resistance prompted us to identify the gene/sresponsible by cloning and sequencing the wild-typegene/s.

As shown in Fig. 1, three genes forming an operon, asin E. coli, were initially identi¢ed by sequence similarity,and two of them (ftsE and X) by complementation anal-ysis of E. coli mutants. This fts gene cluster (ftsY, E andX) is located at 76 min on the E. coli genetic map [17].Recently, clear evidence has been found that ftsE and Xform a complex in the inner membrane that bears thecharacteristics of an ABC-type transporter involved incell division [18]. In addition, a previous publication indi-cates that these genes were conserved as rpoH neighboursin the genome of enteric bacteria [20]; our data and theV. cholerae genome sequence [16] indicate that this extendsto the Vibrionaceae.

The Aeromonas ftsE mutation renders a ¢lamentousphenotype at 37³C, which may interfere with opsonopha-gocytosis, thus explaining the resistance observed in themutant strain. This may be a physical barrier (morpholog-ical problem) for phagocytosis, because opsonisation stillseems to occur, i.e. the amount of C3b deposited is similaramong the wild-type and mutant strains. Reintroductionof the wild-type alleles (ftsE and X) completely restoredthe sensitivity to opsonophagocytosis and the non-¢lamen-tous phenotypes at 37³C. One mechanism to evade phago-cytosis may be simply to grow as aggregates or ¢lamentssu¤ciently large to discourage easy ingestion by phago-cytes, as described for Actinomyces viscous [21] or Derma-tophilus congolensis [22]. Thus a ¢lamentous phenotype at37³C in A. hydrophila ftsE mutants may represent a majoradvance in avoiding an important host defence such asopsonophagocytosis. The Aeromonas ftsE and X geneswere fully able to complement the ¢lamentous phenotypeof both E. coli ftsE mutants, and he ¢lamentous pheno-type was temperature-dependent in opposite ways in bothmutants (Table 3).

Acknowledgements

This work was supported by grants from DGICYT andPlan Nacional de I+D (Ministerio de Educacion y Cul-tura, Spain). L.I. and M.A. are predoctoral fellowshipsfrom the same institution and Generalitat de Catalunya,

Fig. 2. Micrographs of E. coli and A. hydrophila strains grown at di¡er-ent temperatures. E. coli strain RG60 (ftsE mutant) grown at 30³C (A),37³C (B), and the same strain complemented by pACYC-FTS plasmidgrown at 37³C (C). A. hydrophila AH-414 (ftsE mutant) grown at 30³C(D), 37³C (E), and the same strain complemented by pACYC-FTS plas-mid grown at 37³C (F). A clear ¢lamentous phenotype could be ob-served for both strains grown at 37³C, that is restored to a non-¢lamen-tous phenotype by plasmid pACYC-FTS.

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respectively. We thank Maite Polo for her technical assis-tance.

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