Cellular chaining influences biofilm formation and structure in Group A Streptococcus

Names: Jeslyn Poo Shi Ting, Jonas Koh Zhi Xiang, Navin S/O Jeyabalan, Regine Tiong Hui Yi

Background

Group A Streptococcus (GAS), is a human pathogen that causes a variety of human diseases [1,2]. During a GAS infection, the formation of biofilm, which is a complex aggregate of microbes, is an important factor contributing to antibiotic treatment failure [3,4]. It may also play a role in GAS pathogenesis and disease by changing the bacterial transcriptional profile or by modulation of the host response [5-7].

Chaining is a defining morphological feature of GAS, and its contribution to its pathogenicity and biofilm formation has not been deeply investigated. In other bacterial species, chaining contributes to virulence traits like complement evasion and adhesion to host cells, as well as biofilm formation [8-10]. Furthermore, it is poorly understood how GAS regulates its own chain length and it is likely to be complex and highly dependent on environmental conditions.

Previously, a transposon library screen for chain length-influencing genes in S. sanguinis found that a large number of transposon mutants, corresponding to 15% of the genes within the genome, had either a significant increase or decrease in bacterial chain length [11]. This suggests that streptococcal chaining could be a highly regulated process. Growth medium supplemented with homologous antiserum containing anti-M antibodies induces the formation of long chains, while non-immune serum results in chain shortening [12].

In GAS, the autolysin Mur1.2 is predicted to be involved in cell separation and morphology, due to its homology with the peptidoglycan hydrolase LytB in S. pneumoniae [13]. Transcriptomic studies showed that autolysin Mur1.2 is induced by Serine/Threonine kinase Stk, which is an important regulator of cell division, growth and virulence [13]. In addition, the deletion of rgg2, which is a transcriptional regulator, has resulted in overexpression of mur1.2 and increased biofilm formation [14].

In this preprint, the authors investigate how GAS biofilm formation can be directly influenced by host and environmental factors through the modulation of bacterial chain lengths, potentially contributing to persistence and colonization within the host.

Key Findings: 

Overview of the experimental design

Bacterial strains used included both GAS and Escherichia coli (E. coli). Growth conditions were adjusted through the supplementation of the growth medium with lysozyme or serum. The Δmur1.2 deletion mutant was constructed by allelic replacement and complementation of the Δmur1.2 knockout strain was performed by cloning the mur1.2 gene to regulate the chaining of GAS cells.

De-chaining of GAS cells is critical for the formation and stability of a dense biofilm 

The authors demonstrated that the host environment (presence of lysozyme and NaCl) modulates GAS chain length which regulate GAS biofilm morphology and structure. De-chaining of GAS cells (induced by lysozyme as well as addition of non-immune serum) promotes the formation of a denser and compact 3D structure, improving stability of GAS biofilms (Fig. 1). This point was strongly validated with the use of NaCl (Fig. 2) and deletion of mur1.2 (hyperchaining mutant; Fig. 3), both of which increased chaining of GAS cells, decreased biofilm formation characterised by a loose biofilm, with more fragile architecture. Interestingly, this weaker biofilm has reduced antibiotic tolerance.

Figure 1. Lysozyme induces de-chaining of GAS cells of four strains, using negative staining (a), crystal violet staining (b) and confocal microscopy (c,d) Adapted from Figure 1 of Matysik et al. (2020).

Figure 2. NaCl causes high chaining morphology of GAS cells which is attenuated by the addition of lysozyme. Adapted from Figure 2a of Matysik et al. (2020).

Figure 3. Deletion of mur1.2 changes the biofilm structure by producing long chains of GAS cells. Adapted from Figure 3 of Matysik et al. (2020).

 

In vivo analysis of GAS infections on BALB/c mice revealed the host environment influences GAS chaining. 

In addition to the in vitro phenotypes observed for GAS de-chaining in the presence of serum or lysozyme, in vivo validation was sought using the mur1.2 mutant on necrotizing fasciitis mice model. Lesions were harvested and colony forming units were enumerated from both mice inoculated with WT GAS and mur1.2 mutants. There were no significant differences in the colony counts 24 hours post-infection and a microscopic evaluation of lesions through immunostaining showed no major differences from chaining. It is shown that in vivo GAS appears in clumps of cells which is a stark difference compared to the in vitro phenotype in absence of both serum or lysozyme. The lack of chaining seen in vivo is highly suggestive of a dominant host derived factor contributing to GAS morphology. Notably these factors could include but are not limited to, protection for GAS from dechaining factors found in serum through phagocytic cell internalisation or cytosolic concentrations of NaCl that could enabling chaining.

Figure 4. GAS in vivo infection in BALB/c mice via subcutaneous injections. Adapted from Figure 5 of Matysik et al. (2020).

 

What we like about this work:

We like that the authors used a cautious approach to investigate GAS biofilms by applying both in vitro and in vivo models and that they drew on appropriate controls, including de-chaining factors (e.g. lysozyme) in the growth media to simulate a more realistic physiological environment. In addition, the authors opined that classical crystal violet assays may not be the best way to investigate biofilm formation as it does not translate well into in vivo observations. The technical limitations described in this paper will enable future research of GAS biofilm formation and virulence to be more clinically relevant.

 

Future directions/questions for the authors: 

1. The authors have tried to induce augmented GAS chaining with NaCl, and have shown that NaCl can reduce biofilm formation. In the future, authors can repeat this experiment to mimic the natural host (in this case human)’s NaCl concentration variations so as to provide clinical relevance.
2. It will be interesting for the authors to provide opinions and clinical modelling to show how GAS infection can be treated using this paper’s findings (how GAS can be de-chained, and/or how biofilm formation can be disrupted by inhibiting mur1.2 in vivo).

 

References:

 

1. Ralph AP, Carapetis JR. 2013. Group a streptococcal diseases and their global burden. Curr Top Microbiol Immunol 368:1–27.
2. Ferretti JJ, Stevens DL, Fischetti V, Sims Sanyahumbi A, Colquhoun S, Wyber R, Carapetis JR. 2016. Global Disease Burden of Group A Streptococcus. In Ferretti JJ, Stevens DL, Fischetti VA (ed), Streptococcus pyogenes : Basic Biology to Clinical Manifestations. University of Oklahoma Health Sciences Center, Oklahoma City (OK).
3. Baldassarri L, Creti R, Recchia S, Imperi M, Facinelli B, Giovanetti E, Pataracchia M, Alfarone G, Orefici G. 2006. Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation. J Clin Microbiol 44:2721–7.
4. Conley J, Olson ME, Cook LS, Ceri H, Phan V, Davies HD. 2003. Biofilm formation by group a streptococci: is there a relationship with treatment failure? J Clin Microbiol 41:4043–8.
5. Vyas HKN, Proctor EJ, McArthur J, Gorman J, Sanderson-Smith M. 2019. Current understanding of Group A Streptococcal biofilms. Curr Drug Targets doi:10.2174/1389450120666190405095712.
6. Ferretti JJ, Stevens DL, Fischetti VAYoung C, Holder RC, Dubois L, Reid SD. 2016. Streptococcus pyogenes Biofilm. In Ferretti JJ, Stevens DL, Fischetti VA (ed), Streptococcus pyogenes: Basic Biology to Clinical Manifestations, Oklahoma City (OK)
7. Vajjala A, Biswas D, Tay WH, Hanski E, Kline KA. 2019. Streptolysin-induced endoplasmic reticulum stress promotes group A Streptococcal host-associated biofilm formation and necrotising fasciitis. Cell Microbiol 21:e12956.
8. Cho KH, Caparon MG. 2005. Patterns of virulence gene expression differ between biofilm and tissue communities of Streptococcus pyogenes. Molecular Microbiology 57:1545–1556.
9. Dalia AB, Weiser JN. 2011. Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody. Cell Host Microbe 10:486–96.
10. Rodriguez JL, Dalia AB, Weiser JN. 2012. Increased chain length promotes pneumococcal adherence and colonization. Infect Immun 80:3454–9.
11. Evans K, Stone V, Chen L, Ge X, Xu P. 2014. Systematic study of genes influencing cellular chain length in Streptococcus sanguinis. Microbiology 160:307–15.
12. Ekstedt RD, Stollerman GH. 1960. Factors affecting the chain length of Group A Streptococci. Journal of Experimental Medicine 112:687–698.
13. Bugrysheva J, Froehlich BJ, Freiberg JA, Scott JR. 2011. Serine/threonine protein kinase Stk is required for virulence, stress response, and penicillin tolerance in Streptococcus pyogenes. Infect Immun 79:4201–9.
14. Zutkis AA, Anbalagan S, Chaussee MS, Dmitriev AV. 2014. Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes. PLoS One 9:e114784.

 

Posted on: 2 September 2021

doi: https://doi.org/10.1242/prelights.30458

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