FEMS Microbiology Letters 141 (1996) 189-193 ELSEVIER

Constitutive expression of Pasteurella multocida toxin

Isobel C. Hoskins, Alistair J. Lax *

Institute for Animal Health, Compton Laboratory, Compton, Berks. RG20 7NN. UK

Received 18 March 1996; revised 21 May 1996; accepted 28 May 1996

Abstract

The expression of the Pasteurella multocida toxin (PMT) gene toxA was investigated. Growth in vitro at 30°C or added iron caused less than 4-fold repression of toxA expression. The putative repressor TxaR was expressed in Escherichia coli but

deletion and frameshift mutations abolishing TxaR production had no effect on toxA expression. Naturally occurring non- toxigenic mutants which contained the toxA gene had no large rearrangements near toxA or changes in toxA promoter structure. Thus PMT is constitutively expressed and is only regulated in a minor way.

Keywords: Pasteurella multocida toxin; tnaR; Toxin regulation

1. Introduction

The Pasteurella multocida dermonecrotic toxin

(PMT) is the most potent mitogen found for the

Swiss 3T3 cell line, acting at picomolar concentra-

tions to cause maximal DNA replication [l]. In por- cine atrophic rhinitis due to P. multocida, PMT causes nasal turbinate atrophy [2]. The toxA gene

which encodes PMT has been cloned and sequenced

[3-51. It was reported that deletions up to 450 bp 5’

to toxA in a putative repressor gene txaR increased PMT expression S-10-fold in Escherichia coli [5].

Virulence genes are often coordinately expressed in

response to environmental changes [6]; for example, iron concentration [7] or temperature [8]. The aim of this work was to investigate environmental factors which might regulate PMT production, characterise

* Corresponding author. Tel. : +44 (1635) 578 411; Fax: +44 (1635) 577 263; E-mail: lax@bbsrc.ac.uk

naturally occurring toxA mutants and clarify the role of TxaR the putative repressor.

2. Materials and methods

2.1. Strains

E. coli MT102 [5] and DHSa [9] were cultured

using standard methods [9]. DS410 was cultured as

described [lo]. LFB3 is a toxigenic P. multocida

strain [l 11. P. multocida strains 118/l and 118/3 were pig isolates collected in 1988 and 1989. P. mul-

tocida was cultured as previously described [3].

2.2. Plasmid construction

Transformations, DNA preparation and manipu- lation, and sequencing were done by standard meth- ods [9].

pREP2 (Fig. 1) was made from pAJL14 [3] by

0378-1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V PIISO378-1097(96)00220-O

190 I. C. Hoskins, A. J. Lax I FEMS Microbiology Letters 141 (1996) 189-I 93

PAJL16 15.4kb CP P H H P c c UIkb

PAJL14 7.9kb

Fig. 1. Restriction maps of plasmids. The black boxes indicate

the vector DNA. Grey areas indicate the CAT reporter gene-

block. Open boxes indicate the toxA open reading frame and the

large cross-hatches indicate the approximate location of the txaR

open reading frame. Arrows indicate the direction of transcrip-

tion of the CAT and p-lactamase genes. C= &I, P=PstI,

H = HpaI, A = A&II, E = EcoRI, Ev = EcoRV, B = BarnHI. The

ringed PstI site is that which was filled in in pREP3 to create

the frameshift in txaR in pREP4.

deleting the smaller CfuI fragment (to remove up- stream sequences) and replacing the coding region

between the EcoRV and HpaI sites with the chlor- amphenicol acetyltransferase (cat) geneblock (Phar-

macia Ltd). Plasmid pREP3 was constructed from pREP2 by cutting it with AatII and Cl&, isolating

the larger fragment and ligating that to a 2.5-kb &I-HpuI fragment bearing txuR from pAJL15

(Fig. 1). pREP4 was constructed by linearising pREP3 in txuR with PstI, rendering the DNA blunt ended and religating the plasmid. This created a

frameshift in txuR (Fig. 1). pCD12 was made by de-

Table 1

In viva expression of toxA

Sample Viable count* Total countb EBL toxicity

In vitro 1 3 x108 2.8 x 10s 103

In vitro 2 1.5x 10s 1.8~10’ 10s

Pig 1 5.5x 10s 3.2 x 10’ 102

Pig 2 8.8 x 105 2.9 x 10’ 102

“Viable count was determined in duplicate for several dilutions of

cells.

bTotal count was determined as the average count on 20 squares in

the haemocytometer multiplied by the dilution factor.

“EBL toxicity was determined in duplicate. EBL toxicity results

were determined using samples containing the same total cell count

and expressed as the highest dilution at which a cytotoxic effect

was observed on 50% of the cells.

leting the CZuI-BumHI fragment from pAJL14 and religating the plasmid (Fig. 1). Plasmid pSPE716R

was constructed from pSPE525 as described [5].

2.3. PCR

The oligonucleotides 5’-ttacaatggacgctgaagc-3’

and 5’-ggcggaccattcagggg-3’ were prepared. The

100 ~1 PCR reaction in Vent polymerase reaction buffer (New England Biolabs) included 250 ng of

each oligonucleotide, 0.5-l mM MgC12, 0.4 mM dNTPs, approximately 5 ng genomic DNA and 1

unit of Vent polymerase which was added after heat- ing to 94°C (New England Biolabs). The thermal cycle was 94°C for 1 min, 45°C for 1 min, 65°C

for 1 min repeated 35 times.

2.4. Protein analysis

All work involving strains expressing functional PMT or DNA containing the complete toxA gene

was done at ACGM category 3+ containment. Bacteria were harvested, stored and assayed as de-

scribed [ 121 for EBL assays and [ 131 for CAT assays. Minicells were prepared and labelled as described

[lo]. For Western analysis LFB3 cells were grown for at least five generations, harvested at an A600 of 0.5-0.7, lysed in SDS sample buffer [9], and stored

at -20°C in 50% glycerol (v/v). Crude lysates con-

3.3 11 5 6.6

Fig. 2. Western analysis of LFB3 grown under different condi-

tons. Left hand panel: silver stained gel. Right hand panel: ECL

fluorescence. Lanes 1 and 5, 100 PM iron chloride; lanes 2 and

6, no iron addition; lanes 3 and 7, temperature 30°C; lanes 4

and 8, temperature 37°C. The numbers below the right hand

panel represent the relative intensity of the bands measured on a

densitometer against standard amounts of toxin. Equal amounts

of protein were loaded in each lane.

I. C. Hoskins, A. J. Laxl FEMS Microbiology Letters 141 (1996) 189-193 191

taining recombinant PMT were prepared from cells

harvested at Am of 0.8 [12]. Total protein was de- termined using the BCA protein assay kit (Pierce

Chemical Co., USA).

2.5. Western analysis

Proteins were analysed by SDS-polyacrylamide gel

electrophoresis and transferred to Hybond-C mem- brane (Amersham) by standard methods [9]. The membrane was incubated overnight in 10% Marvel,

0.1% Tween-80 in PBS (Tween/PBS) and then in a l/

500 to l/4000 dilution of affinity-purified rabbit poly- clonal antiserum against purified recombinant PMT

in Tween/PBS for 1.5 h. A goat anti-rabbit polyclo- nal antibody (ICN Biochemicals) was used at a l/ 1000 to 114000 dilution in Tween/PBS as the second-

ary antibody. Specific antibody binding was detected by ECL (Amersham), and Kodak Biomax film.

2.6. In vivo growth of P. multocida

Two lZday-old gnotobiotic piglets were pre-

treated with dilute acetic acid and infected with lo8

LFB3 [14]. Two days later bacteria were collected by nasal washing with 10 ml PBS [14], chilled, harvested

and resuspended in PBS. The viable count was de- termined on blood agar plates, and the total cell

count with a haemocytometer. The remaining cells were lysed as described. The piglets were killed 37 days after infection and their turbinates weighed.

3. Results and discussion

3.1. EfSect of environmental factors on PMT

expression

Known environmental triggers of virulence factor expression were tested for their effect on PMT ex- pression in strain LFB3 using Western analysis. Heat shock (41”Q pH (6-8), sodium chloride (0.1-0.4

M), novobiocin (5 @ml), sodium oxalate (5-20 mM, chelates calcium), fetal calf serum (lo%), dipyr- idyl (100 PM, more prevented growth) and growth phase did not affect PMT expression. However, PMT expression was repressed 2-fold at 30°C com- pared to 37°C and 34-fold when 100 pM ferric

A Expression in minicells

12 34

*

‘ne

mol.wt. kDa

- 66 -TxaR

- 45

- 29 - 24 - CAT

B CAT activity 1 jaytrain DJH5alpha 2) #rain MT102

4. :r .&’

%

:z 20

1 c ‘0

4 0

OD at 550nm

Fig. 3. (A) Expression of TxaR in minicells. The products of

minicell labelling were separated by SDS-PAGE, stained and

then exposed to X-ray film. Lane 1, pCDI2; lane 2, pREP2;

lane 3, pREP3; lane 4, pREP4. The TxaR and CAT proteins are

indicated. The protein at 29 kDa is P-lactamase. The protein at

21 kDa corresponds to a truncated PMT product. (B) CAT ac-

tivity of constructs. E. co/i strains bearing either pREP2, pREP3,

or pREP4. The values are the average for two experiments.

Hatched bars denote pREP2, black bars denote pREP3, clear

bars denote pREP4. CAT activity is given in nmol min-’ mg

protein’. Panel 1, E. cofi DHSa host strain. Panel 2, E. coli MT102 host strain.

chloride was added (Fig. 2). A 2-fold increase in PMT expression was previously reported in complex

192 I C. Hoskins, A. J. Lax1 FEMS Microbiohgy Letters 141 (1996) 189-193

medium containing 100 pM dipyridyl [15]. These changes are relatively small compared to those ob-

served with many virulence factors and may not be physiologically relevant [7,8].

PMT production was assessed in vivo in LFB3

recovered from two gnotobiotic pigs 2 days after intranasal inoculation. In vivo samples were lo-fold

less cytotoxic to EBL cells and contained lo-fold less

PMT by Western analysis than in vitro samples re-

presenting the same total bacterial count. However,

the viable count of the in vivo samples was 500-fold

less than the total count (Table 1). The infected pigs showed severe (SO-90%) turbinate loss compared to an uninfected control at post mortem 37 days after infection.

It is impossible to draw conclusions about PMT

expression in vivo from these results. The presence of

large numbers of dead cells is significant as PMT is not exported [ 161 and therefore release from dead

bacteria must be an obligatory part of pathogenesis.

3.2. The role of txaR

The results in this section have been briefly sum-

marised [17]. The role of txuR was investigated with three plasmids pREP2, pREP3, and pREP4 in the E.

coli minicell strain DS410. A protein of 54 kDa cor- responding to TxaR was expressed from pREP3

which contains the complete txaR gene but not from pREP2 or pREP4 (Fig. 3A).

The toxA promoter activity of E. coli DH5a strains bearing either pREP2 or pREP3 was exam-

ined during exponential and stationary phase using

the CAT reporter. In contrast to published observa- tions [5], the average CAT specific activities differed by less than 30% between samples (Fig. 3B). In E.

coli MT102 strains bearing pREP2, pREP3 or pREP4 a maximum difference of 25% in the average CAT specific activities was observed in late exponen- tial phase (Fig. 3B).

To clarify this situation, a published experiment showing repression of toxA expression was repeated. A frameshift mutation at the AvrII site in txaR was constructed in pSPE525 exactly as described [5] to give a duplicate of pSPE716, named pSPE716R. The pSPE716R clones contained 4 bp insertions in txaR as expected. PMT production in each strain

1 2 3 4 5 M

-

-

-

-

mol.

Kd

. 205

116 97

66

45

wt.

Fig. 4. Effect of a frameshift in pPSE525 on toxA expression.

Lane 1, pSPES25; lanes 2-4 pSPE716R clones isolated indepen-

dently; lane 5, purified PMT; lane M, marker proteins. Equal

amounts of total protein were loaded in each lane.

was unchanged by this frameshift (Fig. 4). Therefore TxaR is unlikely to be a repressor of toxA.

3.3. ToxA promoter structure of P. multocida

mutants

Some pathogenic bacteria regulate virulence gene expression via genome rearrangements [ 181. Screen- ing of P. multocida strains by EBL assay and DNA

hybridisation to a 900-bp XbaI fragment from toxA

[19] identified two hybridising isolates from the same herd which were not toxigenic. Crude lysates from these strains were 105-fold less toxic for EBL cells

than LFB3 and polyclonal antibody to PMT de-

tected no cross reacting species. Southern analysis of the mutants with the above DNA fragment re- vealed no gross (> 100 bp) chromosomal changes

near toxA using the enzymes EcoRI, HpaI, HpaII, and ClaI (data not shown). The promoter sequence of PCR amplified DNA up to 200 bp 5’ to the start codon was identical to wild type. These mutants probably contain point mutations or frameshifts in the toxA coding region.

3.4. Conclusions

PMT appears to be expressed constitutively. This has profound implications for animal and also hu-

I. C. Hoskins, A. J. Lax I FEMS Microbiology Letters 141 (I 996) 189-l 93 193

man disease, since toxigenic P. multocida may be

carried by humans [20]. Carriage of toxigenic P. mul-

tocida is likely to lead to exposure to PMT. Analysis of P. multocida recovered from gnotobiotic piglets following infection suggests that the presence and

number of dead bacteria is an important factor in P. multocida infections.

Acknowledgments

We thank Sharon Barnard for her work on natu-

ral PMT mutants and Dr S. Petersen for strains and

plasmids. The work was supported by the Ministry of Agriculture, Fisheries and Food.

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