Previous studies have shown that the membranous ruffles triggered by S
. Typhimurium and the actin-rich comet tails generated during L. monocytogenes
infections share some morphological and structural characteristics with S. flexneri
during their infectious process [6
Typhimurium and L. monocytogenes
recruit and require spectrin cytoskeletal proteins for their efficient invasion as well as for subsequent infectious stages within their host cells [20
]. Based on these similarities, we hypothesized that S. flexneri
may also exploit spectrin cytoskeletal proteins during their infections. Here we have identified important roles for the spectrin cytoskeleton during S. flexneri
initiated macropinocytic invasion of host cells and their presence at comet tails.
During S. flexneri
invasion, a multitude of actin cytoskeletal-associated proteins are recruited to membrane ruffles triggered by T3SS translocated bacterial effectors [6
]. We found that during S. flexneri
infections, p4.1 but not spectrin or adducin, localized to 94% of invasion events. Despite the near complete absence of spectrin or adducin recruitment, when any of the three proteins were disrupted through siRNA treatments, invasion of S. flexneri
was severely decreased. How can the decreased expression of spectrin cytoskeletal proteins that are not markedly recruited to invasion sites have such a dramatic impact on S. flexneri
invasion? Clues to understanding this can be derived from previous research investigating spectrin cytoskeletal involvement during cell migration. There are many shared protein components and structural similarities between S. flexneri
membrane invasion ruffles and membrane protrusions generated during cell migration events. During cell migration, spectrin, adducin and p4.1 often co-localize with, and are necessary for, the recruitment and correct localization of actin-associated machineries to the sub-membranous region of the plasma membrane [14
]. Knockdown of p4.1, or functional perturbation of adducin, both result in an inhibition of membrane protrusions and lack of cell motility [22
]. Thus, it is plausible that proteins involved in actin dynamics leading to the formation of S. flexneri
membrane ruffles and their subsequent invasion are mis-localized when spectrin, adducin, or p4.1 is knocked down. This could explain the observed decrease in bacterial invasion in their absence. Despite not being intensely localized at sites of invasion, we did observe faint recruitment of spectrin and adducin at these invasion sites.
The lack of robust spectrin and adducin recruitment to S. flexneri
invasion sites did not parallel what was found once the bacteria had invaded the host cells, as all three spectrin cytoskeletal components were found surrounding internalized bacteria. We observed their recruitment to invaded bacteria, in the absence of actin, suggesting that those proteins likely arrived at the bacterial interface prior to the recruitment of actin and subsequent comet tail formation. It is possible that spectrin and associated proteins may help recruit the actin machinery to the bacteria, similarly to how they function within lamellipodia [22
], to produce the comet tails enabling intracellular motility. Unfortunately, due to the low abundance of bacteria internalized during spectrin cytoskeletal knockdowns, we were unable to investigate the impact of spectrin cytoskeletal protein involvement in actin recruitment to internalized bacteria.
Upon S. flexneri
generation of full-length actin-rich comet tails, spectrin was found at the comet tails, while p4.1 and adducin were not. Previous work that decorated filamentous actin with the S1 subfragment of myosin identified S. flexneri
comet tails to be dense networks of branched and cross-linked actin filaments [21
]. Cross-linking proteins, such as α-actinin, are recruited to S. flexneri
comet tails and are thought to provide the bacteria with a rigid platform off of which they propel [21
]. Spectrin is an established actin cross-linking protein, increasing the viscosity of actin filaments in vitro
]. This cross-linking characteristic may be at work within S. flexneri
comet tails, however this requires further scrutiny.
As the actin dynamics at the leading edge of motile cells are similar to those occurring during pathogen induced macropinocytotic membrane ruffling and comet tail motility, one would predict that similar components would be present at these sites. L. monocytogenes
and S. flexneri
have been used as model systems to study pseudopodial protrusions for years [27
]. However, the identification of only spectrin and not adducin or p4.1 at fully formed S. flexneri
comet tails, together with the absence of all spectrin cytoskeletal components at L. monocytogenes
comet tails [20
], highlight differences between membrane protrusion events during whole cell motility and those generated by bacterial pathogens. These findings demonstrate the diverse tactics used by microbes to regulate host components and further show that pathogens exploit varying factors during their infectious processes.
Our findings, and findings from other papers (summarized in Additional file 4
: Table S1) demonstrate that not all components of the spectrin cytoskeleton always act in concert. Rather, we have observed that spectrin, adducin, and p4.1 can act in the absence of each other during the pathogenic processes of S. flexneri, L. monocytogenes, S
. Typhimurium and Enteropathogenic E. coli
(EPEC) pathogenesis. Previous studies have highlighted roles for spectrin, adducin and p4.1, irrespective of the influence of one another. Adducin is capable of binding, cross-linking and bundling F-actin, in the absence of spectrin and p4.1 [29
]. Similarily, spectrin is capable of binding actin in the absence of adducin or p4.1 [18
]. Furthermore, purified spectrin and p4.1 can cross-link actin filaments in vitro
, in the absence of adducin [26
]. Adducin is also capable of binding a number of plasma membrane proteins, in the absence of spectrin [30
]. Thus, precedents from other systems support our findings that spectrin, adducin, and p4.1 can act independently during bacterial pathogenesis.