Shigella flexneri Diguanylate Cyclases Regulate Virulence

Abstract

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli , and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-guanosine monophosphate (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. C-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri . In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae , in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome, and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes Importance The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella species is on the rise. Here, we explore how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We find that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella ’s ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri

“…This causes YfiN to remain in an inactivated state, not being able to produce c-di-GMP and affecting various virulence functions in Shigella [ 28 , 31 , 32 , 71 ]. In a similar study regarding the importance of c-di-GMP regulation in Shigella virulence, conducted by Ojha R. et al (2021), it is demonstrated that cloning an active diguanylate cyclase (DGC) gene from Vibrio in S. flexneri elevated the native c-di-GMP levels, which further led to an increase in the biofilm production [ 20 ]. Conversely, DGC Shigella mutant strains also showed altered virulence phenotypes such as reduced biofilm formation and host cell invasion [ 20 ].…”

“…High concentrations of c-di-GMP are known to have a progressive effect on the adhesion and invasion of the bacteria, which eventually aids the pathogen in colonizing the host epithelial cells [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. This relationship between biotic or abiotic surface attachment and c-di-GMP concentration has been previously inspected in the opportunistic human pathogen P. aeruginosa [ 31 , 32 , 74 ] and recently in S. flexneri [ 20 ]. It is known from these studies that on surface contact, there is an upsurge in levels of c-di-GMP, which induces flagella/pilli biosynthesis and surface adherence and boosts virulence [ 22 ].…”

“…S. flexneri, while in the host gastrointestinal tract, encounters various stimuli and pressures such as acidic pH, the presence of bactericidal bile, competition against host microflora, the compulsion of an effective adherence to the epithelial cells to prevent clearance, followed by a successful invasion and evasion from the host immune system [ 10 , 11 , 19 ]. To effectively adapt to these adverse conditions in the host during the infection process, intestinal pathogens such as S. flexneri have developed and adapted various methods to sense and appropriately respond to external stimuli [ 20 ].…”

“…An abundant, highly conserved molecule that acts as a secondary messenger in Shigella and other bacteria facilitating these cellular and molecular adaptations to the host environment is Bis- (3′-5′)-cyclic dimeric GMP (c-di-GMP) [ 20 ]. The rise in c-di-GMP levels inside the bacterial cell is known to promote flagella/pili biosynthesis, motility, biofilm formation, transcription, exopolysaccharide (EPS) production, surface adherence, invasion of host cells, and overall virulence of the bacteria [ 21 , 22 , 23 , 24 , 25 ].…”

Evolution towards small colony variants of pandemic multidrug resistant ST131 Escherichia coli isolates from a 10-year bone infection

“…Not all annotated pseudogenes are necessarily out-of-function, though, as exemplified with the PDE YcgG which consists only of the catalytic EAL domain deleted by its N-terminal signaling domain which can show still, although altered, functionality (Zlatkov and Uhlin 2019). Thus, pseudogenes might have acquired a novel functionality and/or expression pattern (Ojha, Dittmar et al 2021). This genome evolution, including the arisal of pseudogenes leading to the development of small colony variants, on a short time scale resembles, for example, the evolutionary fixed evolution of the pathovar Shigella from commensal E. coli , which occurred on several occasions on an evolutionary short time scale between 270,000 and <400 years ago (The, Thanh et al 2016).…”