are the causative agents of a variety of diseases varying from local infections of the intestinal tract to systemic forms like typhoid fever.1
Typhoid fever is a fatalillness caused by Salmonella
). Worldwide, about 21.7 million cases ofillnesses and 2,17,000 cases of deaths caused by typhoid fever are reported annually.2
South-central Asia and South-east Asia are the regions with high prevalence of typhoid fever (>100/100,000 cases/year).3
The disease is much more severe in Bangladesh, especially in young childrenwhere the S. typhi
infectionis substantially higher.4
The proteomic analysis showed that the crucial steps of pathogenesis of S. typhi
are a result of the production of a particular toxin, the haemolysin,a product of hlyE
Like many otherpore-formingtoxins, the HlyE
toxin is also an important virulence factor among bacteria belonging to the Enterobacteriaceae.6,7
The HlyE, also denoted as ClyA and SheA, belonging to the family cytolysins, forms large, stable pores in target membranes.8
This toxin also causes haemolysis of erythrocytes and has apoptogenic effects on human and murine monocytes/macrophages.9,10
It has been reported that genes coding for close homologues of haemolysin are present in the S.enterica
serovarTyphi or serovarParatyphi A and Shigellaflexneri
genomes.This haemolysin is also required for survival of thebacteria within the host macrophage.11,12
Furthermore, the wild‐type S. typhi
and S. paratyphi
A strains contain functional HlyE proteins, suggesting that the HlyE protein plays important roles in the pathogenesis of these organisms.8
Interestingly, the hlyEgene encoding this potentially toxin protein haemolysinis also present in the non-pathogenic Escherichia coli
K-12 strain, which, however,normally exhibits no haemolytic activity under standard culture conditions.13
This could be due to the fact thatseveral other conditions may influence the regulation of hly
Egene expression in E. coli
In addition to the above findings, the non-haemolytic E. coli
K-12, on the other hand,showed significant hemolytic activity under stress conditions like oxygen and glucose starvation,indicating that stress conditions confer haemolytic phenotype properties upon the non-haemolytic E. coli
In the same study, an underlying molecular mechanism responsible for inducing hly
E gene expression in response to glucose starvation and anaerobic conditions in E. coli
K-12 was also investigated by genetic analyses. Again,the hlyE gene hasalso been found to be present in S. typhi
All the previous findings encouraged the authorsto confirm if the “stress” phenomenon is also applicable to other enteric bacteria like S. typhi.
Therefore, in this study, the haemolytic activity of S. typhi
under environmental stress conditions, like glucose and oxygen starvation, separately and together was investigated.Furthermore,conditions other than stress that induced haemolytic activity were investigated and it was found that recombinant E. coli
,expressing the gene for transcription factor Sly
A (Salmolysin)exhibited high hemolytic activity when cultured in normal conditions without any stresses.
Materials and Methods:
All the organisms used in this study were obtained from the stock culture of the Department of Microbiology, University of Dhaka.The study was conducted during the period of March-December 2015.
New Zealand white rabbits (2-2.5 Kg body weight) were maintained in the Department of Microbiology, University of Dhaka and all experiments using animals were undertaken following ethical issues set by the Faculty of Biological Sciences, University of Dhaka.
Homology analysis of haemolysinof S. typhi and E. coli:
Haemolysin sequence of S. typhi
(NP_805266.1) and E. coli
(AP_001807.1) were obtained from the protein sequence database Genpept (www.ncbi.nlm.nih.gov)and the homology between their amino acid sequences was determined using bioinformatics software tool ClustalW2.
Homology analysis of crp and fnr genes of S. typhi and E. coli:
Sequences of the gene scrp
[NC_003198.1 (4213325..4213957)] and fnr
[NC_003198.1 (1355387..1356181)] of S. typhi
and the sequences of the genes crp
[NC_000913.3 (3486120..3486752)]and fnr
[NC_000913.3 (1398774..1399526)] of E. coli
were obtained from the Gene sequence database (www.ncbi.nlm.nih.gov). The homology analyses of the genes were performed using the bioinformatics software BLASTn.
Culturing S. typhi and E. coli under different conditions:
To observe the haemolytic activity, the S. typhior E. coli
K-12 strains were grown in both normal condition orunder different stress conditions. For normal culture condition, organisms were grown in Luria broth containing 0.2% glucose and incubated aerobically for 24 hours at 37o
C. The stress conditions were appliedby oxygen or glucose starvation, either separately or together.For oxygen starvation condition, both organisms were grown in Luria broth with glucose supplement (0.2%) in sealed bottles and incubated in an anaerobic condition. On the other hand, for the glucose starvation condition, both organisms were grown aerobically but without any glucose supplement. Again, for both glucose and oxygen starvation conditions, both of these organisms were cultured without any glucose supplement in sealed bottlesand incubated anaerobically. All experiments were repeated twice to confirm the reproducibility of the results.
Protein extraction from bacteria:
After 20 h of incubation of S. typhi
or E. coli
K-12 at normal or under different stress conditions, the antibiotic Polymyxin B was added to the media (5μg/ml) and cultures were further incubated for four more hours to break down the cells. Bacterial cultures were then centrifuged at 6,000 rpm for 10 min and the supernatants containing the bacterial proteins were collected.
Assay of haemolytic activity:
The supernatants containing the bacterial proteins were analyzed for haemolytic activity,13
where 0.5ml of 1% rabbitred blood cell (RBC) solution was mixed with 4.5 ml of each of the bacterial proteins extracted fromeither S. typhi
or E. coli
K-12. The tubes werethen incubated for two hours at 37o
C followed by centrifugation at 1,500 rpm for 10 min to pellet the erythrocytes. Absorbancesat 543nm were recorded spectrophotometrically to measure the amount of hemoglobin released into the supernatants.
Cloning of slyAgene:
Isolation of genomic DNA, plasmid DNA and all DNA cloning procedures were carried out following the methods described by Sambrooket al.16
The gene slyA
was amplified by Polymerase Chain Reaction (PCR) (Forward primer 5'-GCGTCAGACATGCATGCTTTAG-3'; Reverse primer 5'-GGTTACTGTCTGTCGACGCTAAACC-3').8
After restriction digestion of both purified PCR product and pACYC184 vector (Cmr) with Sph
I and Sal
I, the insertwas cloned into the dephosphorylated purified vector by cohesive end ligation reaction using DNA Blunting and Ligation Kit (K1512,Fermentas, UK). The successful ligation of sly
A to the vector (pACYC184-slyAvector-insertconstruct) was confirmedby transformation into the chemo-competent E. coli
DH5αcells. Molecular size of the vector-insert constructs isolated from the E. coli
DH5α was confirmed by gel electrophoresis. Upon confirmation, pACYC184-slyA vector-insertconstructswere introduced into the chemo-competent E.coli
K-12 cells and the transformed colonies were selected on chloramphenicol (12.5μg/ml)containing plates.
All data were statistically analyzed using ‘Student’s t-distribution’ to compare the differences in haemolytic activities of the extracted proteins at different culture and stress conditions.
Haemolytic activities of cell extracts of S. typhi
grown under stress conditions like oxygen or glucose starvation, either separately or together, were found to be significantly higher when compared with the normal growth conditions(figure 1).Interestingly,the haemolytic activity was found to be higher in glucose starvation than in oxygen starvation(p<0.001). Again, the hemolytic activity exhibited by S. typhi
was found to be the highest when both of these starvation conditions were applied together than any of the stresses applied alone (p<0.001).
Thestudy also showed that the E. coli
K-12 grown under oxygen and glucose starvation conditions, either alone or together, had similarpattern of hemolytic activitieslike that of S. typhi
grown under the same conditions(figure II).This result also indicates that, under separate oxygen or glucose starvation conditions, S. typhi
showed 1.51 folds and 1.11 folds more hemolytic activities,respectively, when compared with the hemolytic activities of E. coli
K-12 under same conditions. Again, the magnitude of hemolysis was found to be highest in E. coli
K-12, when both oxygen and glucose starvation were applied togther, and the value was comparable to that of the hemolytic activity shown by S. typhi
grown underthe same conditions (figure II). However, the haemolytic activity displayed by E. coli
under oxygen starvation condition was 51% higher and under glucose starvation condition was 11.8% higher than that displayed by S.typhi
under the same conditions.
In addition to the stress factors, other conditions that might induce haemolytic activity in S. typhi
were also investigated. As previously reported, the SlyA
, a transcription factor that positively regulates haemolysin production, is present in both S.typhi
and E. coli
K-12.13,15 To assess the effect of overexpression of SlyA
on induction of haemolytic activity in normal condition, i.e. without any stress, theslyA gene from S. typhi
was isolatedand cloned into E. coli
K-12. The transformed E. coli
was then grownunder normal condition and the degree of haemolytic activity was observed. When the cell extract of transformed cellswas observed for the haemolytic activity, surprisingly a significant degree of haemolysis of RBC was found to occur, when compared with the cell extract of the non-transformed E. coli
The higher haemolytic activities showed by S. typhi at glucose and oxygen starvation conditions (fig.1).
Fig.1: Haemolytic activities of cell extracts of S. typhi grown under normal and stress conditions like oxygen or glucose starvation, either separately or together. Haemolytic activities of the S.typhi grown under stress conditions were found to be significantly higher when compared with the normal condition (p<0.001).
indicated that these particular stresses may be important for turning on the hlyE
gene responsible for hemolysin production in S. typhi
that ultimately caused hemolysis. This result was in accordance with the findings of Westermerk et al14
where the non-hemolytic E. coli
K-12 also showed significant hemolytic activity under stress conditions.
Again, the comparison of the haemolytic activities of S. typhi
and E. coli
Fig.2: Comparison of haemolytic activities of cell extracts ofS. typhi with that of E. coli K-12 grown under normal and stress conditions like oxygen or glucose starvation, either separately or together. Under separate oxygen or glucose starvation conditions, S. typhi showed more hemolytic activities when compared with the hemolytic activities of E. coli K-12 under same conditions (p<0.001).
clearly indicated that the haemolytic activities induced in S. typhi
under stress conditions is quite consistent to our predictions, since S. typhi
also harbors hlyE
gene like that of E. coli
K-12. Though the hlyE
gene is present in E. coli
K-12, however, it is silent in normal culture condition and its expression can be induced through stress conditions. It has been reported that two members of the cAMP Receptor Protein (CRP) family of transcription factors control the expression of hlyE
in E. coli
K-12, where CRP enhances hlyE
expression in response to glucose starvation and Fumarate Nitrate Reduction (FNR) regulatory protein enhances hlyE
expression in response to oxygen starvation.15
In this context, using bioinformatics analysis, in this study it was observed that the genes crp and fnr of transcription factors CRP and FNR respectively, wereboth found in S. typhi
and these two genes of S. typhi
were 88% identical with the crp and fnr genes of E. coli
K-12, respectively. It was also observed that both S. typhi
and E. coli
K-12 contain functional homologs of hlyE
and the protein encoded by the S. typhi
is 90% identical in amino acid sequence to that of the hlyE
of E. coli
K-12. In reality, the S. typhi
does not express this gene in normal culture conditions like that of E. coli
K-12.Therefore, it is evident from the current study that S. typhi
has similar strict control mechanisms for the hlyE
gene and expression of hlyE
gene can be induced in S. typhi
under oxygen and/or glucose starvation conditions that confer the hemolytic ability upon S. typhi.
This novel finding clearly indicates that environmen-tal starvation conditions may act as stress factors for the induction of the hlyE
gene to produce hemolysin in S. typhi.
Also, the increased haemolytic activity of E. coli
K-12 transformed with the transcription factor SlyA
gene from S. typhi
(fig.3). clearly indicated that overproduction of SlyA
in E. coli
K-12 is independent of any culture condition. The result also suggests that the hemolytic activity can be induced in a different bacterial strain when the gene for transcription factor SlyA
was cloned from another bacterial strain. This finding supports the phenomenon that SlyA
overproduction antagonizes the negative effects of the regulatory protein H-NS, a nucleoid structuring protein that represses hlyE
Fig.3: Comparison of hemolytic activities of cell extracts of non–transformed E. coli K-12 and slyA gene transformed E. coli K-12 grown under normal culture condition (Oxygen+Glucose). SlyA gene transformed E. coli K-12 showed higher degree of hemolytic activity compared to the hemolytic activity shown by the non-transformed E. coli K-12 (p<0.001).
Based on this findings of molecular mechanisms, we assessed the degree of hemolysis seen in the hemolytic assay with RBC, after overexpression of slyA
gene under normal culture conditions, and all results indicated that hlyE
gene might be expressed in response to appropriate environmental signals.
Haemolytic activity can be considered to be one kind of virulence factor.17
Therefore, findings of this study are very significant in clinical perspectives, since stress conditions may induce virulent properties in bacterial strains. There is evidence that stresses like starvation, acidic pH and heat shock may induce the expression of some virulence genes.18
Again, it has been suggested that conditions stimulating the production of the haemolysin might be encountered during infection by ,i>E. coli0.001).> strains.13
As both S. typhi
and E. coli
are enteric bacteria, therefore, it is assumed that these organisms may encounter oxygen starvation condition in intestinal environment which may induce the production of haemolysin in the host. This is again supported by the report that anaerobiosis has been shown to induce the invasion phenotype in Salmonella.19-21
Hence, during the course of their infection, these bacteria may encounter anaerobic condition or limited glucose, which may induce haemolysin production. Therefore, all our results along with reports of other investigators clearly indicate that, S. typhi
may exhibit hemolytic activity under glucose or/and oxygen starvation conditions. Again, the SlyA
transcription factor may also induce haemolysin production, if it is overexpressed in vivo, without going through any starvation conditions.
Findings of this study may also help to understand how Salmonella
survive in the macrophages and how the haemolysin may help S. typhi
in causing infections as a crucial virulence factor. Therefore, the haemolysin protein can be a potential antigenic target for development of a vaccine as an alternative therapeutic agent for Salmonella
infections. Previously, in Bangladesh, studies on anti-HlyE responses in patients have been carried out with a view to developing improved diagnostic assays.22
In this novel study, it was assayed production of HlyE
toxin under various conditions in order to analyse this protein with a view to developing a novel vaccine against this toxin in future in Bangladesh.
This project has been partially funded by University Grants Commission (UGC), Bangladesh.We would like to heartily thank Dr. Siraje Arif Mahmud for his expert assistance with this manuscript.
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