Phase Variation of LPS and Capsule Is Responsible for Stochastic Biofilm Formation in Francisella tularensis

Abstract

Biofilms have been established as an important lifestyle for bacteria in nature as these structured communities often enable survivability and persistence in a multitude of environments. Francisella tularensis is a facultative intracellular Gram-negative bacterium found throughout much of the northern hemisphere. However, biofilm formation remains understudied and poorly understood in F. tularensis as non-substantial biofilms are typically observed in vitro by the clinically relevant subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica (Type A and B, respectively). Herein, we report conditions under which robust biofilm development was observed in a stochastic, but reproducible manner in Type A and B isolates. The frequency at which biofilm was observed increased temporally and appeared switch-like as progeny from the initial biofilm quickly formed biofilm in a predictable manner regardless of time or propagation with fresh media. The Type B isolates used for this study were found to more readily switch on biofilm formation than Type A isolates. Additionally, pH was found to function as an environmental checkpoint for biofilm initiation independently of the heritable cellular switch. Multiple colony morphologies were observed in biofilm positive cultures leading to the identification of a particular subset of grey variants that constitutively produce biofilm. Further, we found that constitutive biofilm forming isolates delay the onset of a viable non-culturable state. In this study, we demonstrate that a robust biofilm can be developed by clinically relevant F. tularensis isolates, provide a mechanism for biofilm initiation and examine the potential role of biofilm formation.

Why vary what’s working? Phase variation and biofilm formation in Francisella tularensis

“…Highlighting the differences in biofilm formation among F. tularensis subspecies, O-Ag mutants in F. novicida form biofilms that are equal or less than the parental wild-type (Champion et al, 2019). Mlynek et al identified a link between variation of the O-Ag and biofilm formation in F. tularensis (Mlynek et al, 2021). In agreement with the Champion findings, biofilm formation typically required at least 7 days to occur.…”

“…Francisella species have been shown to form biofilm [reviewed by van Hoek (2013) ], though clinically important subspecies of F. tularensis tend to form a less defined biofilm with sparse cell density than F. novicida or other Francisella species( Margolis et al, 2010 ; Mahajan et al, 2011 ). Recent studies demonstrate that O-antigen (O-Ag) of the LPS can influence the biofilm-forming capacity of Type A and B isolates, changing this perception of biofilm formation in F. tularensis ( Champion et al, 2019 ; Mlynek et al, 2021 ). This review aims to explore where phase variation and biofilm may play a role in the survival and pathogenesis of F. tularensis which could lead to new therapies to prevent disease.…”

“…LPS is considered a major virulence factor in F. tularensis as it allows the bacterial cell to evade the host immune response ( Sandstrom et al, 1992 ; Gunn and Ernst, 2007 ; Wang et al, 2007 ; Weiss et al, 2007 ) and deleterious mutations within genes involved in LPS or O-Ag synthesis often lead to attenuation ( Raynaud et al, 2007 ; Apicella et al, 2010 ; Kim et al, 2012 ; Jones et al, 2014 ; Rasmussen et al, 2014 , 2015 ; Chance et al, 2017 ). While the structure of F. tularensis LPS contains notable and unique features for lipid A, core, and O-Ag [reviewed by Gunn and Ernst, 2007 ], recent studies suggest the O-Ag is a driving determinant of biofilm-forming capacity ( Champion et al, 2019 ; Mlynek et al, 2021 ). As the case with a wide range of other Gram-negative bacteria, O-Ag has been demonstrated to play a role in biofilm formation ( Nakao et al, 2006 , 2012 ; Murphy et al, 2014 ; Hathroubi et al, 2016 ).…”

“…Mlynek et al identified a link between variation of the O-Ag and biofilm formation in F. tularensis ( Mlynek et al, 2021 ). In agreement with the Champion findings, biofilm formation typically required at least 7 days to occur.…”

“…It was subsequently determined that biofilm formation was associated with a distinct population of gray variants that emerged within the culture. While the exact alteration of the LPS in these variants was unknown, western blotting with α-LPS or α-capsule mAbs against O-Ag yielded no reactivity ( Mlynek et al, 2021 ). The studies Champion et al and Mlynek et al found that external conditions, such as culture medium and pH, impacted biofilm development, which suggests additional gene regulation and/or environmental checkpoints beyond antigenic variation of O-Ag may factor into F. tularensis biofilm formation.…”