Abstract: Acid Tolerance Response (ATR) in Shigella sonnei and Shigella flexneri was induced by pre-exposing the mid exponential phase cultures at sub lethal pH of 4.5 for 90 min before challenging them in lethal pH of 3.0. The ATR was exhibited by both species with S. flexneri displaying a higher tolerance to lethal acid killing. Analyses of protein profiles of both unadapted and adapted cells using 2-dimensional gel electrophoresis (2D-PAGE) revealed that the acid adapted cells of S. sonnei demonstrated differential expression of 19 Acid Stress Proteins (ASPs). Five of these proteins were synthesized de novo, 11 were upregulated while three other proteins were repressed. In S. flexneri, two proteins were synthesized de novo, four upregulated and two proteins were repressed. Two of these proteins with estimated molecular weights of 14.1 and 15.1 kDa and isoelectric points (pI) of 5.50 and 5.58, respectively were found to be consistently induced in both species. An inducible ATR in Shigella may protect the bacteria from environmental pH stress and aid in their pathogenesis. The detection of proteins important for ATR may present novel targets for antimicrobial intervention.
INTRODUCTION
Shigella is the primary causative agents of shigellosis, a bacillary dysentery which is transmitted directly through faecal-oral routes or indirectly through faecal-contaminated food and water. They are neutrophiles thus the bacteria will need to first overcome the acid stress mechanisms employed by the human body before they can establish an infection and disseminate. These include the acidic environment of the stomach, with pH value as low as 2.0 (Verdu et al., 1994), the volatile fatty acids in the small intestine with pH around 4.0-4.5 (Gahan and Hill, 1999) and the acidic interior of phagolysosomes (Oh et al., 1996; Rathman et al., 1996). However, studies have shown that microorganisms have the ability to adapt to environmental stress. Microbial cells if first pre-exposed or adapted to sub-lethal dose of acid will subsequently survive better when challenged to lethal dose as compared to the unadapted cells. Such phenomenon of inducible ATR has been observed in a number of enteric pathogens (Foster and Hall, 1991; Goodson and Rowbury, 1989; Karem et al., 1994; Murphy et al., 2003; Tiwari et al., 2004). Nevertheless, most of these studies were done on the stationary phase of the bacteria cells where cells would naturally induce a general stress response that provides resistance against most environmental challenges.
Few studies have been conducted on actively growing exponential phase cells which lack the general stress response. Enteric pathogens in contaminated foods and drinks can potentially be in an exponential phase of growth. Most food are acidic, either naturally or due to added preservatives, thus providing a favorable environment for adaptation of the microorganisms which upon consumption may protect them from the acid stress environment in the stomach, small intestines and phagocytes. Moreover, the inducible ATR also involves changes in the gene expression of the cells (Audia et al., 2001; Foster and Hall, 1990) whereby, the synthesis of some cellular proteins are upregulated or repressed while some are synthesized de novo as part of the survival mechanism to protect the cells from subsequent damages.
The aims of the present study were to determine whether ATR could be induced in both S. sonnei and S. flexneri and to investigate the differences in their tolerance response. Although, a closely related study has been documented whereby the survival of both Shigella species in acidic fruit juices was determined (Bagamboula et al., 2002), the present study took a different approach by using inorganic acid instead. The synthesis ASPs during the adaptation period would also be observed which may further serve as novel cellular targets for the development of new antimicrobial compounds.
MATERIALS AND METHODS
Both S. sonnei S37 and S. flexneri S307 were clinical isolates obtained from the Institute of Medical Research (IMR) Kuala Lumpur and the cultures were maintained on Nutrient Agar (NA) at 37°C.
Preparation of mid-exponential cells culture: One loopful of a 24 h culture was inoculated into 100 mL of NB pH 7.0 and grown at150 rpm, 37°C for 24 h. The cultures were then diluted (1:50) in fresh NB and further grown to mid exponential phase (OD600 of 0.05). The cells were harvested, washed twice in NB by centrifugation at 7,000 rpm for 10 min and the pellets were resuspended in 10 mL NB at pH 7.0. These cell suspensions (approx 107 cells mL‾1) were used in all subsequent experiments as mid-exponential cell cultures.
Acid sensitivity of Shigella sonnei and Shigella flexneri: The sensitivity of both Shigella species to different acid concentrations was studied to determine their lethal and sub-lethal pH values. The lethal acid pH is referred to the external pH value that instantly killed and reduced the viability of the bacterial population to less than 1% within the 3 h exposure while the sub-lethal pH is defined as the pH that inhibits growth and reduced the viability of the cells by 30-50% at the end of the exposure period.
Mid-exponential cells were inoculated into a series of flasks containing 100 mL NB and adjusted to an initial OD600 of 0.05. One milliliter sample was taken immediately, diluted in PBS and plated to determine the initial viable count. The pH of each culture was then adjusted with HCl. Following that, the cultures were incubated at 150 rpm, 37°C. Samples were then taken every 30 min and the cell count was determined.
Induction of ATR: The ability of S. sonnei and S. flexneri to induce tolerance to lethal acid was carried out by first pre-exposing the mid-exponential phase cells of both species to a sub-lethal acid pH value for 90 min (equivalent to approximately two doublings) before challenging them to the lethal pH value.
The adapted cells of the Shigella species were prepared by exposing the mid-exponential cells to the sub-lethal pH 5.0 at 37°C, 150 rpm for 90 min while, the unadapted cells remained at pH 7.0. The initial cell count (t0) was determined at the end of the exposure period before the adapted cells were challenged to lethal pH of 3.0 to test for acid tolerance. A sample of the cells was immediately taken (t1) and the cultures were further incubated with samples taken at every 10 min over a period of 40 min. Each experiment was performed in three repeats.
Two-dimensional SDS-PAGE: The acid adapted and unadapted cells were harvested separately and washed twice in PBS and sterile distilled water. A volume of 50 μL of the packed cells were lysed in lysis buffer (8 M urea, 2% CHAPS, 0.002% bromophenol blue, 0.1% protease inhibitor solution (Sigma, USA) using a sonicator. (Sartorius, Germany). The resulting lysate was centrifuged at 13,000 rpm, 5°C to remove cell debris and the clarified protein sample was immediately used or stored at -80°C. Protein concentrations were determined using a Bicinchoninic acid protein assay kit (Sigma, USA) according to the manufacturer’s instructions.
An aliquot in of each cell lysate containing 15 μg of proteins were resolved using two-dimensional SDS-PAGE (ZOOM Benchtop Proteomic System, Invitrogen, USA) according to the manufacturer’s instructions. The isoelectric focusing was performed on a broad range strip of pH 4-7 using a protocol of 175 V for 15 min, followed by voltage ramping from 175-2000 V in 45 min and a final 2000 V for 30 min. The second dimension PAGE was performed on a pre-cast Bis-Tris 4-12% gradient gel at 200 V for 50 min. The gels were then stained using Invitrogen’s SilverQuest silver staining kit and analysed using Imaxia’s Gelfox software.
The 2D proteins profiles of S. sonnei and S. flexneri were first compared to a partially annotated 2D gel image of S. flexneri 2a strain 2457T (Liao et al., 2003). Several clear matching protein spots were chosen as landmark and used to estimate the relative density, Molecular Weight (MW) and pI values of other spots in the gels. The Gelfox software was used to compare the proteins profiles between unadapted and acid adapted cells of both species. These proteins were identified preliminarily by comparing their MW and pI values to virtual protein spots on an in silico 2D gel image of S. flexneri 2457T (ARGONNE National Lab (USA) Gelbank, http://gelbank.anl.gov(Last time access on this date 2015-05-31)). For each protein spot of interest, corresponding spots within a range of ±5% of the MW and ±0.1 pI units from the in silico virtual gel were chosen. Preliminary identification of the protein spots was done using the database at the website. The list was then filtered based on the potential of these proteins in contributing towards stress mechanism, excluding hypothetical proteins or other proteins without known functions.
RESULTS AND DISCUSSIONS
Acid sensitivity and ATR of Shigella sonnei and Shigella flexneri: Both mid-exponential cells of S. sonnei and S. flexneri were rapidly killed at pH below 4.0 (Fig. 1a-b). Approximately 30 and 70% of the S. sonnei populations remained viable after 3 h at pH 4.5 and 5.0, respectively. Shigella flexneri appeared to have a higher acid resistant and able to grow normally at pH 5.0 while about 40% of the cells survived at pH 4.5. Hence, pH 3.0 and 4.4 was chosen as the lethal and sub-lethal pH, respectively for both species.
Gorden and Small (1993) reported that most Shigella species were resistant to extreme acid conditions as they were able to survive exposure to pH 2.5 for at least two hours. However, in the present study, both cells of S. sonnei and S. flexneri were unable to survive at pH lower than 3.5 where the cells died almost instantly. The difference could probably be due to the different phase of the cultures used.
| Fig. 1(a-b): | Mid-exponential cells of (a) Shigella sonnei and (b) Shigella flexneri grown in various pH. Cells were sampled at 30 min interval for 3 h to determine viable cell counts |
Studies have shown that resistance was observed when the bacteria reached at least the late-exponential phase with full expression of acid resistance expressed in the stationary phase (Benjamin and Datta, 1995; Gorden and Small, 1993). The acid survival ability in stationary phase of S. flexneri was reported to be associated with the sigma factor σs encoded by rpoS (Nunoshiba and Demple, 1994; Waterman and Small, 1996). In contrast, the mid-exponential phase used in the present study is a phase where cells were actively growing due to the abundance of nutrients in the environment. Such condition will unlikely to induce any general resistance system thus these cells are less resistant when exposed to extreme acid conditions. Shigella flexneri also appear to have a better ability to survive in acidic conditions. This could possibly due to ability of the S. flexneri membrane to block extracellular protons from entering the cells, more efficient buffering capacity or a better proton-pump system to discharge extra protons outside the cells.
Both S. sonnei and S. flexneri displayed an inducible ATR as shown in Fig. 2a-b. Adapted cells of both strains survived better as compared to the corresponding unadapted cells (p<0.05) at every sampling time point. Shigella flexneri cells displayed a stronger ATR, with higher survival percentages as compared to cells of S. sonnei at all time points (p<0.05). After 40 min of acid challenge, 1% of the adapted S. flexneri cells remained viable which is 250,000-fold higher than the unadapted cells (p<0.01). However, at this similar point, only about 0.005% of the adapted S. sonnei cells remained viable which is 700-fold higher than the unadapated cells (p<0.01). At the end of the 90 min exposure the number of viable declined to similar levels in both adapted cells and unadapted cells. Thus, the ATR conferred by adaptation is only able to protect the adapted cells effectively for the first half-hour of the acid challenge. A stronger ATR observed in S. flexneri which could be due to a greater tolerance towards acid. While, most studies were done on stationary phase cells, this is probably the first attempt to compare the ATR of these pathogenic Shigella species in a complex medium at mid-exponential phase.
| Fig. 2(a-b): | Induction of ATR in Shigella at pH 4.5. Mid-exponential cells of (a) Shigella sonnei and (b) Shigella flexneri were adapted by pre-exposing to a sub-lethal pH of 4.5 for 90 min and challenged at a lethal pH of 3.0. Error bars indicate the standard errors of the means |
2-D gel analysis: In S. sonnei, a total of 139 matched spots were detected from both gels while the unmatched spots were not included in the analysis (Fig. 3a-b). The density data revealed that 19 of these spots showed at least a two-fold increase or decrease in their concentrations in the adapted cells which could also be verified by visual comparison of their density. Of these, five were novel proteins expressed only in the adapted cells while the expression of 11 proteins was induced or over-expressed and the three remaining proteins were repressed in these cells. In S. flexneri, 134 protein spots could be matched the (Fig. 4a-b) whereby, eight of these were differentially expressed in the adapted cells which consist of two novels, four induced and two repressed proteins.
By comparing the MW and pI of protein spots to a 2D virtual gel, a few possible ASPs candidate were identified. It is interesting to note that two of the novel proteins spots found in acid adapted cells of S. sonnei (Fig. 3a, spots 4 and 5) were also synthesized de novo in acid adapted cells of S. flexneri (Fig. 4a, spots 1 and 2), with predicted Molecular Weights (MW) of 14.1 and 15.1 kDa and isoelectric points (pI) of 5.50 and 5.58, respectively. Thus, these proteins are probably strong candidates involved in ATR mechanism of Shigella as they were present in adapted cells of both species. Preliminary identification showed strong possibility of the first protein being TolR, an inner membrane protein, while the second one could be either osmotically induced protein OsmC, a heat shock protein ibpA or a membrane bound ATP synthase, AtpC.
Two systems were found to be involved in acid resistance of stationary phase Shigella grown in minimal medium (Bearson et al., 1997). However, recent studies using E. coli K-12 mutants revealed that none of these systems are involved in the development of acid tolerance in exponential phase cells grown in rich medium (Seputiene et al., 2006). This suggests a different survival mechanisms are involved in both exponential and stationary cells of E. coli which could also be the case in Shigella as they are closely related.
| Fig. 3(a-b): | The 2D gel of total protein extracts from (a) Acid adapted and (b) Unadapted Shigella sonnei cells. The rectangles in (a) indicate proteins synthesized de novo, circles indicate over-expressed proteins while arrows indicate repressed proteins as compared to the unadapted cells |
The involvement of the inner membrane protein TolR has also been speculated in other studies. In S. typhimurium, the acid adapted cells were found to be capable of altering the outer membrane protein profile of the bacteria (Leyer and Johnson, 1993) while, studies on E. coli cells suggest that modification of the lipid bilayer in cell membrane helps to decrease the proton permeability thus protecting the cells from acid shock (Chang and Cronan, 1999). This is supported by Booth et al. (2002), who suggested that adaptive response to acid is responsible in changing the membrane permeability. Tol R is one of the proteins known to be responsible in maintaining the structural integrity of the outer membrane of bacteria cells (Lazzaroni et al., 1999). It is postulated here that Shigella could employ a similar passive mechanism of modifying the cell membrane during exposure to mild acid thus decreasing the proton permeability, making it more resistant to extreme acidic conditions.
A second passive mechanism that could be employed is by increasing the buffering capacity towards excessive H+ by cellular constituents (Hill et al., 1995). Some bacteria may have specialized systems in the cell membrane which are activated upon adaptation to mild acidic conditions (Seputiene et al., 2006). These include the induction of proteins which bind external protons more effectively or help H+ ions to access the sites of binding inside the cell membrane or cytoplasm thus contributing to the acid resistance.
| Fig. 4(a-b): | The 2D gel of total protein extracts from (a) Acid adapted and (b) Unadapted Shigella flexneri cells. The rectangles in (a) indicate proteins synthesized de novo, circles indicate over-expressed proteins while arrows indicate repressed proteins as compared to the unadapted cells |
In the present study, the induction of AtpC, an ATP synthase protein in ATR could probably involved in transporting the excess H+ in these species. In L. monocytogenes, the synthesis of sub-unit b of ATP synthase was also speculated to play a similar role in removing extra H+ from the cytoplasm (Phan-Thanh et al., 2000).
A possible role for exponential phase ATR in Shigella infections: Shigella is noted for their uniquely small infectious dose whereby only 10-500 cells are sufficient to cause infection which is attributed to the general stress response. The primary natural habitat for Shigella is the human gut. Once expelled from the human body, the bacteria is postulated to enter into stationary phase where it remains ‘dormant’ until transmitted to another suitable host possibly via contamination. Food contaminated with Shigella could be acidic and able to initiate growth under favorable conditions and enters the exponential phase. Hence, the involvement of the ATR mechanism that allows survival in acid could be a significant factor in their ability to cause infections. Clinically, S. flexneri causes a more severe diarrhea compared to S. sonnei. It is possible that a stronger ATR mechanism in S. flexneri contribute towards their virulence by allowing larger numbers of cells to survive in the intestinal environment. However, a direct link between ATR and virulence in this species remains to be established.
ACKNOWLEDGMENT
The work was supported by a grant from the Malaysian Ministry of Higher Education Fundamental Research Grant Scheme (RMI/ST/FRGS 5/3/FST(6/2008)).