Invasion of HeLa Cells by Shigella Species Clinical Isolates Recovered from Pediatric Diarrhea

Abstract

Shigella is considered a major public health concern, especially for children younger than 5 years of age in developing countries. The pathogenicity of Shigella is a complex process that involves the interplay of multiple genes located on a large, unstable virulence plasmid as well as chromosomal pathogenicity islands. Since various factors (including virulence and antibiotic resistance genes) are associated with the severity and duration of shigellosis, in this article, we aim to evaluate whether the invasion of HeLa cells is affected by Shigella spp. isolates with different characteristics (including serogroups, virulence gene profiles, and antibiotic resistance patterns) recovered from pediatric patients in Tehran, Iran. Cell invasion ability of 10 Shigella isolates with different serogroups (Shigella flexneri and Shigella sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin) were measured by plaque-forming assay in HeLa cell lines. The results show that all the selected Shigella spp. isolates recovered from pediatric patients were able to invade HeLa cells, but the total number and average size of plaques were different between the isolates. The higher invasion ability of S. flexneri isolates in HeLa cells compared to S. sonnei isolates was attributed to the presence of particular virulence genes; however, the role of each of these virulence factors remains to be determined.

Introduction

S higella species continue to be a major health issue globally, with an estimated burden of 80–165 million episodes and 600,000 deaths annually, particularly in children younger than 5 years of age in developing countries (Francois Watkins and Appiah, 2020; WHO, 2022). The genus Shigella is divided into four species (Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii), among which S. flexneri and S. sonnei accounted for the most common causes of shigellosis in developing countries, including Iran (Ewing, 1949; Moosavian et al., 2019; Sadredinamin et al., 2022; Talukder et al., 1999; Yaghoubi et al., 2017). Shigella spp. is responsible for various clinical manifestations, from gastroenteritis to bacillary dysentery (Aslam and Okafor, 2022). The associated virulence factors of Shigella spp. are located both on the chromosomal pathogenicity islands (PAIs) and a large virulence plasmid named pINV (Schroeder and Hilbi, 2008). Unstable PAIs are mobile elements that come in various combinations based on the Shigella spp. and subtype (Schmidt and Hensel, 2004; Schroeder and Hilbi, 2008). The plasmid encodes the type III secretion system (T3SS), ipa, ipg, and vir genes playing an important role in tissue invasion and the intracellular lifestyle of Shigella spp. The plasmid also mediates other indications like watery diarrhea through Shigella enterotoxin-2 (ShET2) encoded by the sen gene (Karimi-Yazdi et al., 2020; Mattock and Blocker, 2017).

Upon ingestion of contaminated food, Shigella translocate through M cells, invade macrophages, and induce apoptosis (Ashida et al., 2014; Aslam and Okafor, 2022). After being released from the dead macrophages, Shigella penetrates the basolateral side of the colonic epithelium by injecting virulence factors via its T3SS. This invasion process is highly dependent on the presence of Shigella IpaB, IpaC, and IpaD. Once inside the epithelium, the pathogen multiplies and disseminates to neighboring cells through actin polymerization, facilitated by the virulence factors IcsA and T3SS effector protein IpgD (Carayol and Van Nhieu, 2013; Köseoğlu et al., 2022). The T3SS also secretes various effector proteins, including VirA, which play essential roles in all stages of the infection, from the initial uptake to cell-to-cell spread (Germane et al., 2008).

These events result in inflammation and destruction of the colonic epithelium, which eventually leads to the clinical manifestations of diarrhea (sometimes containing blood and mucus) and could be accompanied by abdominal cramps, and fever, seizures, sepsis, hemolytic-uremic syndrome, and postreactive arthritis are less common complications depending on the Shigella spp. (Francois Watkins and Appiah, 2020; WHO, 2005).

The invasion of gastrointestinal epithelia is a critical virulence mechanism employed by several enteric pathogens including Shigella spp., which can vary among different isolates (Willer Eda et al., 2004; Zhang et al., 2017). This mechanism can be effectively simulated in vitro using mammalian cell lines, such as Hep-2 or HeLa cells (Rahman et al., 2002). Accordingly, this study aimed to investigate the invasion of HeLa cells by selected Shigella spp. isolated from pediatric diarrhea in Tehran, Iran.

Materials and Methods

Clinical samples

Considering the importance of Shigella as the main cause of death in children due to dysentery (Ndungo et al., 2022), a total of 10 Shigella strains were chosen according to the serogroups (S. flexneri and S. sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin), which were determined in our previous study (Tables 1 –3) (Karimi-Yazdi et al., 2020). This study was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences (code: IR.SBMU.MSP.REC.1398.262).

Table 1.

List of Selected Shigella spp. Isolates Along with Their Virulence Genes Determination, Antibiotic Resistance Pattern, and Invasion of HeLa Cells

Strain	Virulence genes pattern	Antibiotic resistance pattern	Numbers of plaque	Average size of plaques (mm)	Plaque efficiency
SS1	ipaH, virA, sen	CFM, CTX, NA, AMP, SXT	96	1.62	0.192
SS2	ipaH, ipaD	MN, NA, SXT	202	0.5	0.404
SS3	ipaH, ipaB, ipaC, ipgD	CFM, CTX, NA, AMP, SXT, MN, AZM	18	0.77	0.036
SS4	ipaH, ipaC, sen, virA	MN, NA, SXT	15	0.17	0.03
SS5	ipaH, ipaC	MN, NA, SXT	13	0.59	0.026
SF1	ipaH, ipaB, ipaC, ipaD, virA	AMP, SXT, NA	134	2.04	0.274
SF2	ipaH, ipaB, ipaC, ipaD, ipgD, sen, virA	—	294	0.3	0.588
SF3	ipaH, ipaB, ipaC, ipaD, ipgD, sen, virA	AMP, SXT, NA	123	0.18	0.246
SF4	ipaH, ipaB, ipaC, ipaD, ipgD, sen, virA	AMP, SXT, CTX, CFM, AZM	138	0.25	0.276
SF5	ipaH, ipaB, ipaC, virA	AMP, SXT, CTX, CFM, MN, NA	23	0.31	0.046
ATCC 12022	ipaH, ipaB, ipaC, ipaD, ipgD, sen, virA	—	29	0.36	0.058

AMP, ampicillin; ATCC, American Type Culture Collection; AZM, azithromycin; CFM, cefixime; CIP, ciprofloxacin; CTX, cefotaxime; LEV, levofloxacin; MN, minocycline; NA, nalidixic acid; SF, Shigella flexneri; SS, Shigella sonnei; SXT, trimethoprim-sulfamethoxazole.

Table 2.

Shigella spp. Virulence Genes Determination and Invasion of Hela Cells

Virulence genes
Strains	ipaH	ipaB	ipaC	ipaD	ipgD	sen	virA	Plaque efficiency
SS1	+	_	_	_	_	+	+	0.192
SS2	+	_	_	+	_	_	_	0.404
SS3	+	+	+	_	+	_	_	0.036
SS4	+	_	+	_	_	+	+	0.03
SS5	+	_	+	_	_	_	_	0.026
SF1	+	+	+	+	_	_	+	0.274
SF2	+	+	+	+	+	+	+	0.588
SF3	+	+	+	+	+	+	+	0.246
SF4	+	+	+	+	+	+	+	0.276
SF5	+	+	+	_	_	_	+	0.046
ATCC	+	+	+	+	+	+	+	0.058

ATCC, American Type Culture Collection; SF, Shigella flexneri; SS, Shigella sonnei.

Table 3.

Shigella spp. Antibiotic Resistance Pattern and Invasion of HeLa Cells

Strains	Antibiotic									Plaque efficiency
Strains	SXT	NA	AMP	MN	CTX	CFM	AZM	CIP	LEV	Plaque efficiency
SS1	R	R	R	S	R	R	S	S	S	0.192
SS2	R	R	S	R	S	S	S	S	S	0.404
SS3	R	R	R	R	R	R	R	S	S	0.036
SS4	R	R	S	R	S	S	S	S	S	0.03
SS5	R	R	S	R	S	S	S	S	S	0.026
SF1	R	R	R	S	S	S	S	S	S	0.274
SF2	S	S	S	S	S	S	S	S	S	0.588
SF3	R	R	R	S	S	S	S	S	S	0.246
SF4	R	S	R	S	R	R	R	S	S	0.276
SF5	R	R	R	R	R	R	S	S	S	0.046
ATCC	S	S	S	S	S	S	S	S	S	0.058

AMP, ampicillin; ATCC, American Type Culture Collection; AZM, azithromycin; CFM, cefixime; CIP, ciprofloxacin; CTX, cefotaxime; LEV, levofloxacin; MN, minocycline; NA, nalidixic acid; R, Resistance; S, Sensitive; SF, Shigella flexneri; SS, Shigella sonnei; SXT, trimethoprim-sulfamethoxazole.

Cell culture

Shigella preparation and tissue culture

For the plaque-forming assay, bacterial strains were streaked onto Tryptic Soy Agar with 0.01% (w/v) Congo red. After incubation at 37°C overnight, red colonies were selected and incubated in Luria-Bertani at 37°C overnight. For the assay, overnight bacterial cultures were subcultured and grown to a mid-exponential phase (OD₆₃₀ reading of 0.6–0.9) and diluted before use (1:16,000).

HeLa cells were maintained and grown in Roswell Park Memorial Institute (RPMI) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin. Cell cultures were maintained at 37°C with 5% CO₂ for growth. Three to five days before the bacterial plaque-forming assay, HeLa cells were seeded at 500,000/well into 6-well plates and allowed to grow confluent.

Plaque-forming assay

Briefly, HeLa cells were grown to confluence in 6-well plates and washed with phosphate-buffered saline sequentially before infection with Shigella (5 × 10⁴ colony-forming units [CFUs]/mL). Sixty minutes after infection (RPMI, 5% [v/v] FBS, 20 μg/mL) gentamycin was added to each well. Afterward, Wright-Giemsa dye (5%) was added 48 h after infection for 30 s. Plaque images were taken after washing with distilled water using an inverted microscope camera. The size and number of plaques were measured using ImageJ software (Koestler et al., 2018b).

Shigella flexneri American Type Culture Collection (ATCC) 12022 and Escherichia coli ATCC 25922 were used as positive and negative controls, respectively.

The invasion was calculated from the plaque-forming unit according to the following formula (Ranallo et al., 2010): $P l a q u e e f f i c i e n c y = n u m b e r o f p l a q u e s ∕ n u m b e r o f i n o c u l a t e d b a c t e r i a \times 100$

Results

The invasion assay showed that all the tested isolates were able to invade the HeLa cells after an hour, while the total number and average size of plaques were different between the strains (Table 1). The total number of plaques counted among all isolates was 1056 (with an average of 105.6 for each strain). According to the results, the number of S. flexneri and S. sonnei plaques was 712 (with an average of 142.4 for each strain) and 344 (with an average of 68.8 for each strain), respectively. Strain SF2 (S. flexneri) produced the highest number of plaques in HeLa cells with 294 plaques; however, strain SS5 (S. sonnei) had the lowest plaque-forming power with 13 plaques compared to E. coli ATCC 25922 as a negative control (Fig. 1).

FIG. 1.

Plaques of the SF2 (A) and SS5 (B) isolates formed in HeLa cell line after an hour incubation at 37°C. SF, Shigella flexneri; SS, Shigella sonnei.

The average sum of plaque areas of S. flexneri and S. sonnei strains was 3.08 mm (on average of each strain of S. flexneri, 0.61 mm) and 65.3 mm (on average of each strain of S. sonnei, 0.73 mm), respectively.

Discussion

The invasion and dissemination into intestinal epithelial cells are pivotal steps in the Shigella spp. infection cycle (Carayol and Van Nhieu, 2013; Zhu et al., 2021). Our understanding of these processes is largely based on the plaque-forming assay that can be determined in vitro using mammalian cells with successful interaction, such as Henle intestinal epithelial cells or HeLa cells (Sansonetti et al., 1986; Sharma and Puhar, 2019). Accordingly, we compared the invasion of Shigella spp. isolates with that of the reference strains and found that all isolates were capable of invading HeLa cells. These results are relatively similar to those of the study by Zhang et al. They reported that all 12 investigated S. flexneri isolates demonstrated invasive phenotype to HeLa cells (Zhang et al., 2013).

However, based on 16 Shigella strains, Omidi et al. concluded that 11 strains had invasive properties in HEP-2 cell culture (Omidi et al., 2017). The method used in their study was different from this research, which probably explains why the results were not in line with each other.

Compared with S. sonnei strains, S. flexneri strains were able to invade a greater range of HeLa cells, showing their possible ability to invade more host cells. Almost identical plaques were formed by S. sonnei and S. flexneri strains, indicating that both species have nearly the same ability to multiply between intestinal epithelial cells. Three of the four bacteria that formed the least number of plaques (<25 plaques) were related to S. sonnei (75%). All four mentioned strains lacked the ipaD gene, and three strains lacking the ipgD gene were also obtained. IpaD is a scaffold protein that localize the tip of the type III secretory apparatus, has a supporting role in IpaB, and is crucial for Shigella invasion (Barta et al., 2012). Mitochondrial destruction by IpaD results in macrophage death, which occurs in conjunction with pyroptosis by IpaB (Arizmendi et al., 2016). IpgD is another effector protein that is associated with Shigella's efficient entry upon contact with epithelial cells. A lack of IpgD interferes with the early stage of infection through the effect on membrane and cytoskeletal rearrangements (Koestler et al., 2018a; Mattock and Blocker, 2017; Niebuhr et al., 2000). As a result, it can be assumed that a smaller number of plaques might be charged with a lack of ipaD and/or ipgD genes.

Among the examined isolates, SF2 and SS5 samples produced the highest and lowest number of plaques in HeLa cells, respectively, compared to the negative control. The results showed that SF2 possessed all investigated genes; however, SS5 carries only ipaC and ipaH genes. According to the fact that Shigella virulence genes are required for efficient bacteria invasion, reduced virulence genes can explain the small number of plaque-forming cells in the SS5 sample (Mattock and Blocker, 2017).

The current experiment was performed 1 h after incubation, while it is suggested to follow up the invasion of Shigella spp. in a longer time for the complete destruction of the cell layers by Shigella spp.

Conclusion

This study characterized the ability of S. flexneri and S. sonnei isolates with different virulence features to invade HeLa cell monolayers. Specifically, we found that S. flexneri isolates, exhibited higher invasion capacity in HeLa cell lines than S. sonnei isolates. Moreover, the invasion of HeLa cells by Shigella serogroups was higher among isolates harboring specific virulence genes (ipaD and/or ipgD); however, to arrive at a definitive conclusion, further studies with a larger sample size are required.

Footnotes

Acknowledgments

We would like to express our sincere thanks to members of the Departments of Microbiology and Immunology at the Shahid Beheshti University of Medical Sciences in Tehran, Iran.

Authors' Contributions

M.S. and M.K.-Y. provided project administration and wrote the article. Z.G. collected the strain, worked on concept and design of the study and critically revised the article. M.S., M.K.-Y. and M.T. analyzed and interpreted data. Z.G., M.T. and G.E. critically revised the article. All authors approved the final version of the article and the authorship list.

Disclosure Statement

No competing financial interests exist.

Funding Information

This scientific research was granted by Shahid Beheshti University of Medical Sciences, Tehran, Iran (grant no.: 14809). All authors are primarily involved in education or medical research and are not directly supported by the government.

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