Investigating the Role of Cytoskeletal Dynamics in Cronobacter Invasion: A Study of Caco-2 and H4 Cell Lines
Abstract
1. Introduction
2. Materials and Methods
2.1. Bacterial Isolates
2.2. Human Cell Lines
2.3. Eukaryotic Cytoskeleton Inhibitors
2.4. Invasion Experiments
2.5. Investigating the Role of Inhibitors in Bacterial Invasion
2.6. Statistical Analysis
3. Results
3.1. Bacterial Invasion
3.2. Effect of Inhibitors on Bacterial Invasion of H4 Cell Line
3.3. Effect of Inhibitors on Bacterial Invasion of Caco-2 Cell Line
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
Appendix A
NTU No | Species | ST | Country | Source | Year | Disease | Site of Isolation |
---|---|---|---|---|---|---|---|
701 | C. sakazakii | 4 | France | Clinical | 1994 | Fatal neonatal infection, NECIII | Infant, peritoneal fluid |
709 | C. sakazakii | 4 | France | Clinical | 1994 | Septicaemia | Infant trachea isolate |
767 | C. sakazakii | 4 | France | Clinical | 1994 | Fatal neonatal meningitis | Infant trachea isolate |
1569 | C. malonaticus | 307 | USA | Clinical | 1994 | Fatal infant meningitis | Blood isolate |
939 | E. coli K1 | 95 | UK | Clinical | NA | Clinical | Enteral Feeding Tube |
358 | Salmonella enteritidis 358 | NA | Positive control in tissue culture experiments | ||||
1230 | E. coli K12 | NA | Negative control in tissue culture experiments |
Isolate | Cell Line | Without Inhibitor (CFU/mL) | CytoD (CFU/mL) | Vin (CFU/mL) | Colch (CFU/mL) | Tax (CFU/mL) | Noco (CFU/mL) |
---|---|---|---|---|---|---|---|
C. sakazakii 701 | H4 | 1.5 × 105 | 3.25 × 104 | 3.15 × 103 | 1.32 × 104 | 1.43 × 105 | 1.32 × 105 |
Caco-2 | 5.77 × 104 | 8.17 × 104 | 2.92 × 104 | 3.96 × 104 | 3.10 × 104 | 3.39 × 104 | |
C. sakazakii 709 | H4 | 1.64 × 105 | 4.53 × 104 | 3.86 × 104 | 1.44 × 104 | 1.40 × 105 | 2.33 × 105 |
Caco-2 | 1.23 × 105 | 5.84 × 105 | 2.79 × 105 | 3.05 × 105 | 2.45 × 104 | 4.89 × 104 | |
C. sakazakii 767 | H4 | 2.29 × 105 | 5.8 × 104 | 8.36 × 103 | 1.83 × 104 | 3.71 × 105 | 2.69 × 105 |
Caco-2 | 2.18 × 105 | 6.84 × 105 | 3.69 × 105 | 3.36 × 105 | 3.31 × 104 | 1.19 × 105 | |
C. malonaticus 1569 | H4 | 3.3 × 105 | 4.87 × 104 | 3.94 × 104 | 1.79 × 104 | 3.11 × 105 | 2.77 × 105 |
Caco-2 | 3.66 × 105 | 6.26 × 105 | 2.31 × 105 | 4.38 × 105 | 1.44 × 105 | 2.67 × 105 | |
E.coli 939 | H4 | 8.78 × 103 | 4.61 × 103 | 3.51 × 102 | 3.10 × 103 | 4.44 × 103 | 2.81 × 103 |
Caco-2 | 8.22 × 103 | 3.79 × 103 | 4.46 × 103 | 4.22 × 103 | 1.23 × 104 | 6.58 × 103 | |
Salmonell enteritidis 358 | H4 | 2.40 × 106 | 1.4 × 106 | 5.03 × 105 | 1.11 × 106 | 1.58 × 106 | 1.27 × 106 |
Caco-2 | 7.29 × 105 | 7.96 × 105 | 5.62 × 105 | 4.57 × 105 | 3.01 × 105 | 3.54 × 105 |
References
- Colonne, P.M.; Winchell, C.G.; Voth, D.E. Hijacking host cell highways: Manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens. Front. Cell. Infect. Microbiol. 2016, 6, 107. [Google Scholar] [CrossRef]
- Sarkar, P.; Kontsedalov, S.; Lebedev, G.; Ghanim, M. The actin cytoskeleton mediates transmission of “Candidatus Liberibacter solanacearum” by the Carrot Psyllid. Appl. Environ. Microbiol. 2021, 87, e02393-20. [Google Scholar] [CrossRef] [PubMed]
- Liu, X.; Xu, H.; Feng, Y.; Hou, X. Development of anticancer drugs: From mechanical properties of tumor stiffness to cytoskeleton-targeting natural products. Preprints 2021. [Google Scholar] [CrossRef]
- Kim, M.; Song, K.; Jin, E.J.; Sonn, J. Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way. Exp. Mol. Med. 2012, 44, 521–528. [Google Scholar] [CrossRef] [PubMed]
- Lambert, C.; Schmidt, K.; Karger, M.; Stadler, M.; Stradal, T.E.B.; Rottner, K. Cytochalasans and their impact on actin filament remodeling. Biomolecules 2023, 13, 1247. [Google Scholar] [CrossRef]
- Laisne, M.-C.; Michallet, S.; Lafanechère, L. Characterization of microtubule destabilizing drugs: A quantitative cell-based assay that bridges the gap between Tubulin based- and cytotoxicity assays. Cancers 2021, 13, 5226. [Google Scholar] [CrossRef]
- Meyer, D.H.; Rose, J.E.; Lippmann, J.E.; Fives-Taylor, P.M. Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans. Infect. Immun. 1999, 67, 6518–6525. [Google Scholar] [CrossRef]
- Naydenov, N.G.; Marino-Melendez, A.; Campellone, K.G.; Ivanov, A.I. Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal. BioEssays 2024, 46, 2400160. [Google Scholar] [CrossRef]
- de Souza Santos, M.; Orth, K. Subversion of the cytoskeleton by intracellular bacteria: Lessons from Listeria, Salmonella and Vibrio. Cell. Microbiol. 2015, 17, 164–173. [Google Scholar] [CrossRef]
- Navarro-Garcia, F.; Serapio-Palacios, A.; Ugalde-Silva, P.; Tapia-Pastrana, G.; Chavez-Dueñas, L. Actin cytoskeleton manipulation by effector proteins secreted by diarrheagenic Escherichia coli pathotypes. BioMed Res. Int. 2013, 2013, 374395. [Google Scholar] [CrossRef]
- Mohan Nair, M.K.; Venkitanarayanan, K. Role of bacterial OmpA and host cytoskeleton in the invasion of human intestinal epithelial cells by Enterobacter sakazakii. Pediatr. Res. 2007, 62, 664–669. [Google Scholar] [CrossRef]
- Healy, B.; Cooney, S.; O’Brien, S.; Iversen, C.; Whyte, P.; Nally, J.; Callanan, J.J.; Fanning, S. Cronobacter (Enterobacter sakazakii): An opportunistic foodborne pathogen. Foodborne Pathog. Dis. 2010, 7, 339–350. [Google Scholar] [CrossRef]
- Hariri, S.; Joseph, S.; Forsythe, S.J. Cronobacter sakazakii ST4 strains and neonatal meningitis, United States. Emerg. Infect. Dis. 2013, 19, 175–177. [Google Scholar] [CrossRef] [PubMed]
- Ibrahim, K.M.; Alsonosi, A.M.; Agena, M.B.; Elgamoudi, B.A.; Forsythe, S.J. Multiplex determination of K-antigen and colanic acid capsule variants of Cronobacter sakazakii. Genes 2024, 15, 1282. [Google Scholar] [CrossRef] [PubMed]
- Alsonosi, A.M.; Ibrahim, K.M.; Elgamoudi, B.A.; Agena, M.B.; Forsythe, S.J. The potential role of rpoS and ompR in the acid resistance and desiccation tolerance of Cronobacter malonaticus Strains. Microbiol. Res. 2025, 16, 53. [Google Scholar] [CrossRef]
- Holý, O.; Cruz-Córdova, A.; Xicohtencatl-Cortes, J.; Hochel, I.; Parra-Flores, J.; Petrželová, J.; Fačevicová, K.; Forsythe, S.; Alsonosi, A. Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples. Microb. Pathog. 2019, 127, 250–256. [Google Scholar] [CrossRef] [PubMed]
- Alsonosi, A.; Hariri, S.; Kajsík, M.; Oriešková, M.; Hanulík, V.; Röderová, M.; Petrželová, J.; Kollárová, H.; Drahovská, H.; Forsythe, S.; et al. The speciation and genotyping of Cronobacter isolates from hospitalised patients. Eur. J. Clin. Microbiol. Infect. Dis. 2015, 34, 1979–1988. [Google Scholar] [CrossRef]
- Joseph, S.; Sonbol, H.; Hariri, S.; Desai, P.; McClelland, M.; Forsythe, S.J. Diversity of the Cronobacter genus as revealed by multilocus sequence typing. J. Clin. Microbiol. 2012, 50, 3031–3039. [Google Scholar] [CrossRef]
- Joseph, S.; Desai, P.; Ji, Y.; Cummings, C.A.; Shih, R.; Degoricija, L.; Rico, A.; Brzoska, P.; Hamby, S.E.; Masood, N.; et al. Comparative analysis of genome sequences covering the seven Cronobacter species. PLoS ONE 2012, 7, e49455. [Google Scholar] [CrossRef]
- Law, R.J.; Gur-Arie, L.; Rosenshine, I.; Finlay, B.B. In vitro and in vivo model systems for studying enteropathogenic Escherichia coli infections. Cold Spring Harb. Perspect. Med. 2013, 3, a009977. [Google Scholar] [CrossRef]
- Buhrke, T.; Lengler, I.; Lampen, A. Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2. Dev. Growth Differ. 2011, 53, 411–426. [Google Scholar] [CrossRef]
- Huang, X.; Huang, C. Fructose shields human colorectal cancer cells from hypoxia-induced necroptosis. Npj Sci. Food 2024, 8, 71. [Google Scholar] [CrossRef]
- Alsonosi, A.M.; Holy, O.; Forsythe, S.J. Characterization of the pathogenicity of clinical Cronobacter malonaticus strains based on the tissue culture investigations. Antonie Van Leeuwenhoek 2018, 112, 435–450. [Google Scholar] [CrossRef]
- Claud, E.C.; Savidge, T.; Walker, W.A. Modulation of human intestinal epithelial cell IL-8 secretion by human milk factors. Pediatr. Res. 2003, 53, 419–425. [Google Scholar] [CrossRef] [PubMed]
- Agena, M.B. Neonatal Exposure to Pathogens: Determining Key Virulence Factors. Ph.D. Thesis, Nottingham Trent University, Nottingham, UK, 2017. Available online: https://irep.ntu.ac.uk/id/eprint/32859 (accessed on 18 August 2025).
- Lněničková, K.; Šadibolová, M.; Matoušková, P.; Szotáková, B.; Skálová, L.; Boušová, I. The modulation of phase II drug-metabolizing enzymes in proliferating and differentiated CaCo-2 cells by hop-derived prenylflavonoids. Nutrients 2020, 12, 2138. [Google Scholar] [CrossRef]
- Liu, L.; Wang, Z.; Park, H.G.; Xu, C.; Lawrence, P.; Su, X.; Wijendran, V.; Walker, W.A.; Kothapalli, K.S.; Brenna, J.T. Human fetal intestinal epithelial cells metabolize and incorporate branched chain fatty acids in a structure specific manner. Prostaglandins Leukot. Essent. Fat. Acids 2017, 116, 32–39. [Google Scholar] [CrossRef]
- Uapipatanakul, B. A study of the effects of waste egg and shrimp shells on the toxicity immobilisation of chemicals. Orient. J. Chem. 2018, 34, 1926–1929. [Google Scholar] [CrossRef]
- Miles, A.A.; Misra, S.S.; Irwin, J.O. The estimation of the bactericidal power of the blood. J. Hyg. 1938, 38, 732–749. [Google Scholar] [CrossRef] [PubMed]
- Sun, L.; Yang, S.; Deng, Q.; Dong, K.; Li, Y.; Wu, S.; Huang, R. Salmonella Effector SpvB disrupts Intestinal epithelial barrier integrity for bacterial translocation. Front. Cell. Infect. Microbiol. 2020, 10, 606541. [Google Scholar] [CrossRef]
- Rudrabhatla, R.S.; Selvaraj, S.K.; Prasadarao, N.V. Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells. Microbes Infect. 2006, 8, 460–469. [Google Scholar] [CrossRef]
- Tsugawa, H.; Ono, T.; Murakami, H.; Okawa, Y. Invasive phenotype and apoptosis induction of Plesiomonas shigelloides P-1 strain to Caco-2 cells. J. Appl. Microbiol. 2005, 99, 1435–1443. [Google Scholar] [CrossRef]
- Mittal, R.; Grati, M.; Gerring, R.; Blackwelder, P.; Yan, D.; Li, J.D.; Liu, X.Z. In vitro interaction of Pseudomonas aeruginosa with human middle ear epithelial cells. PLoS ONE 2014, 9, e91885. [Google Scholar] [CrossRef] [PubMed]
- Alkeskas, A.; Ogrodzki, P.; Saad, M.; Masood, N.; Rhoma, N.R.; Moore, K.; Farbos, A.; Paszkiewicz, K.; Forsythe, S. The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes. BMC Infect. Dis. 2015, 15, 449. [Google Scholar] [CrossRef] [PubMed]
- Kim, K.P.; Loessner, M.J. Enterobacter sakazakii invasion in human intestinal Caco-2 cells requires the host cell cytoskeleton and is enhanced by disruption of tight junction. Infect. Immun. 2008, 76, 562–570. [Google Scholar] [CrossRef] [PubMed]
- Ayibieke, A.; Wajima, T.; Kano, S.; Chatterjee, N.S.; Hamabata, T. The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion. Microb. Pathog. 2024, 190, 106636. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Agena, M.B.; Ibrahim, K.M.; Alsonosi, A.M.; Saad, M.T.; Elgamoudi, B.A. Investigating the Role of Cytoskeletal Dynamics in Cronobacter Invasion: A Study of Caco-2 and H4 Cell Lines. Appl. Microbiol. 2025, 5, 89. https://doi.org/10.3390/applmicrobiol5030089
Agena MB, Ibrahim KM, Alsonosi AM, Saad MT, Elgamoudi BA. Investigating the Role of Cytoskeletal Dynamics in Cronobacter Invasion: A Study of Caco-2 and H4 Cell Lines. Applied Microbiology. 2025; 5(3):89. https://doi.org/10.3390/applmicrobiol5030089
Chicago/Turabian StyleAgena, Mahmoud B., Khaled M. Ibrahim, Abdlrhman M. Alsonosi, Mohamed T. Saad, and Bassam A. Elgamoudi. 2025. "Investigating the Role of Cytoskeletal Dynamics in Cronobacter Invasion: A Study of Caco-2 and H4 Cell Lines" Applied Microbiology 5, no. 3: 89. https://doi.org/10.3390/applmicrobiol5030089
APA StyleAgena, M. B., Ibrahim, K. M., Alsonosi, A. M., Saad, M. T., & Elgamoudi, B. A. (2025). Investigating the Role of Cytoskeletal Dynamics in Cronobacter Invasion: A Study of Caco-2 and H4 Cell Lines. Applied Microbiology, 5(3), 89. https://doi.org/10.3390/applmicrobiol5030089