Upon entry into the host cell, Lm is known to modify its transcriptional program ^{4– 7} . The transcription factor positive regulatory factor A (PrfA) is a master transcriptional activator of genes necessary for Lm pathogenesis, including prfA itself ^{8, 9} . The expression of prfA is regulated by a variety of cues, allowing Lm to adapt to different environments. PrfA translation is known to be dependent on temperature, with higher translation levels at 37°C compared to 30°C ¹⁰ . Recently, it was found that the scarcity of branched-chain amino acids, as would be encountered by Lm during infection, leads to upregulation of prfA transcription ^{11, 12} . Furthermore, glutathione, abundant within the host cytosol, has been uncovered as an allosteric activator of PrfA protein activity ^{13, 14} . In addition to activating PrfA, glutathione was discovered to covalently attach to a conserved cysteine on LLO ¹⁵ . This S-glutathionylation abolishes LLO hemolytic activity, but the precise mechanism by which this reversible post-translational modification affects infection is unknown.

L-glutamine, abundant within host blood plasma and host cell cytosol, has recently been reported as another major cytosolic cue for the upregulation of virulence genes in Lm ¹⁶ . It is currently unknown whether L-glutamine, similarly to glutathione, affects PrfA activity at the post-translational level.

To further investigate the cues sensed by Lm for the regulation of virulence, a screen was performed to identify Lm genes required for expression of the surface protein ActA ¹⁷ . Interestingly, most of the genes important for ActA expression are implicated in bacterial redox homeostasis. Since the host cell can induce oxidative stress as a means of antibacterial activity, redox changes may serve as another cue for the regulation of virulence genes in Lm.

The discovery of novel environmental cues sensed by Lm will continue to be important for the study of the infectious process. Indeed, earlier studies have shown an intracellular upregulation of some virulence factors, e.g. InlK or LntA, but the exact cues were not elucidated ^{18, 19} . Interestingly, other virulence factors such as InlJ or LLS are not expressed in cultured cells but are upregulated in vivo either in the liver and blood ²⁰ or within the intestine ²¹ , again upon undefined environmental cues. Thus, further work is required to determine what currently uncharacterized signals may be sensed by Lm for the upregulation and activation of virulence genes that are poorly expressed in vitro.

It has long been known that actin polymerization drives Lm host cell entry ²² as well as intracellular and intercellular motility ²³ . Once Lm reaches the host cytosol, ActA is transcriptionally upregulated and localized to the bacterial cell surface, where it recruits and activates the host actin regulator the actin-related protein 2/3 (Arp2/3) complex ²⁴ . The resulting actin cloud surrounding the bacteria enables it to evade detection by the host autophagy machinery ^{25– 27} . ActA polarization at one of the bacterial cell poles results in a polarized polymerization of actin. The resulting actin “comet tails” propel the bacteria within the host cell cytosol and facilitate cell-to-cell spread. Although this process is well characterized, recent results have uncovered novel insights into the composition of the host Arp2/3 complex, how the actin cytoskeleton is involved in intracellular and intercellular motility, and how these processes are dependent on the stage of infection and subcellular context.

Exploiting the Arp2/3 complex during infection. The Arp2/3 complex is composed of seven subunits: the Arp2 and Arp3 proteins and five Arp complex proteins (ARPC1–5) ^{28– 32} . When activated by nucleation-promoting factors, it binds to a pre-existing actin filament and catalyzes the formation of a de novo Y-branched actin filament. Interestingly, Lm ActA mimics host nucleation-promoting factors to recruit and activate Arp2/3 near the bacterial surface ³² .

We recently discovered differential requirements for subunits of the Arp2/3 complex for distinct aspects of Lm infection that require actin, i.e. entry and actin-based motility ³³ . Strikingly, ARPC1B, but not ARPC1A, appears to be critical for efficient Lm cell invasion. In contrast, ARPC1A, but not ARPC1B, is required for actin comet tail formation. Together, these results suggest that different isoforms of ARPC1 are exploited by Lm differently. Both ARPC4 and ARPC5 appear to be dispensable for cell invasion. In contrast, ARPC5 is not critical for actin tail formation. Thus, rather than existing as a single canonical complex, different Arp2/3 complexes may be formed by different subunits, and this modularity can be exploited by Lm for distinct steps of infection ³³ . The mechanism by which Lm can activate different Arp2/3 complexes and the effect of differential Arp2/3 activation on the actin cytoskeleton are still unknown.

It is currently unclear whether different Arp2/3 complexes exist and play a role in vivo. Nevertheless, the existence of different Arp2/3 complexes has also been recently reported in the case of focal adhesions ³⁴ and the actin-driven intracellular propulsion of vaccinia virus ³⁵ . The recent discovery of sick but living human children with frameshift mutations in ARPC1B, the predominant ARPC1 isoform expressed in blood cells ³⁶ , suggests critical but distinct roles for different components of the Arp2/3 complex in vivo.

Moving inside cells: mechanisms of Lm intracellular propulsion. Actin polymerization is known to propel intracellular Lm, but the precise mechanism of force generation has remained unclear. There are two prevailing models for actin polymerization-dependent intracellular propulsion of Lm. In the “Brownian ratchet” model, growing tangential actin filaments protrude and provide the propulsive force ³⁷ . The alternate “macroscale elastic propulsion” model implicates large-scale deformation of the actin meshwork as propelling the bacterium forward ³⁷ . Whether Lm intracellular propulsion is driven by individual actin filament elongation or by elasticity of the actin network was unclear.

Recent cryo-electron tomography of Lm-associated actin comet tails both within the cell ³⁸ and within cell-free extracts ³⁹ has shed some light on this process. The network of actin comet tails is composed of both branched and, surprisingly, some bundled filaments ³⁹ . The novel discoveries of additional F-actin bundles throughout the comet tail perpendicular to the direction of motion ³⁸ in addition to tangentially orientated filaments to the bacterial surface suggest that elastic propulsion is the major driving force of Lm propulsion.

These studies are reminiscent of the debate concerning the lamellipodial actin network in migrating cells. The canonical view of Arp2/3-mediated branched actin networks of lamellipodia ^{28– 31} was challenged by a report implicating very little branched actin but instead many overlapping parallel actin bundles ⁴⁰ . The suggestion that actin filaments were mainly unbranched in lamellipodia was controversial ^{41– 43} . Ultimately, a consensus was reached: lamellipodia are once again considered to contain Arp2/3-mediated branches of actin, but there are far fewer of them than expected ⁴⁴ . Membrane-tethered actin polymerizers are thought to mechanically and transiently link actin protrusions to the leading edge plasma membrane of a migratory cell ³⁷ . This transient F-actin polymerization model is similar to the model of actin propulsion of Lm ³⁷ . Altogether, these recent studies highlight the fruitful collaboration of studies of F-actin polymerization in cell migration and Lm propulsion.

In addition to actin comet tails, Lm also induces actin-based bacterial protrusions at the host cell plasma membrane to drive cell-to-cell spread. The actin network in Lm-mediated protrusions is composed of parallel actin filaments ³⁸ —more parallel and less branched than would be expected for Arp2/3-driven polymerization. The Rho-family GTPase cell division cycle protein 42 (Cdc42) is a conserved upstream regulator of host nucleation-promoting factors and Arp2/3 ^{45, 46} but has no role in Lm actin comet tail formation ⁴⁷ . In polarized epithelial tissue culture, Lm actin-based protrusions must counteract cortical tension. Lm partially relieves this tension by secreting the protein InlC, which inhibits Tuba, a Cdc42 activator ^{48, 49} . While these results suggest that Cdc42 activity restricts Lm cell-to-cell spread, a subsequent report by another group suggests that membrane protrusion formation requires active Cdc42 and the actin regulator formin ⁵⁰ , which induce bundled F-actin. The reason for the conflicting requirements of Cdc42 activity for Lm cell-to-cell spread is unclear, although the authors speculate that the discrepancy may be because of the difference in cell types used (polarized epithelial Caco-2 versus non-polarized HeLa cells). Further work is required to ascertain the different requirements for Cdc42 activity in Lm intercellular spread and how the choice of model tissue culture affects these requirements.

Recently, new host cell factors that are recruited to the Lm comet tail were discovered. In addition to the known ActA targets Arp2/3 and enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) ^{51– 54} , ActA was recently shown to recruit lamellipodin. Lamellipodin is a binding partner of Ena/VASP and an actin regulator in lamellipodia ⁵⁵ that promotes Lm cell-to-cell spread ⁵⁶ . Interestingly, lamellipodin is recruited to Lm actin comet tails independently of Ena/VASP, highlighting that lamellipodin can bind to F-actin. Although lamellipodin promotes cell-to-cell spread, curiously, lamellipodin knockdown increased the speed of actin-propelled Lm ⁵⁶ . How lamellipodin both promotes Lm intercellular spread and appears to reduce Lm comet tail speed remains to be clarified. Another group has found that lamellipodin can bind directly to F-actin independently of Ena/VASP in vitro and in cultured migratory cells, possibly promoting lamellipodial formation ⁵⁵ . Together, these results highlight the subversion of host cell lamellipodial formation by Lm to induce cell-to-cell spread.

Actomyosin contractility and Lm. Non-muscle myosin II (myosin) is an actin-based motor protein that assembles into bipolar filaments to exert contractile forces. Interestingly, myosin is known to inhibit Lm infection ⁴⁸ . As mentioned above, suppression of Cdc42 activity in polarized epithelial cells favors cell-to-cell spread, presumably through relaxation of cortical tension ^{48, 49} . However, direct quantification of the relaxation of cortical tension by Lm is lacking, and it would be interesting to measure tension as routinely performed in developmental biology research ^{57, 58} . In addition, pharmacological inhibition of myosin was shown to favor Lm host cell adhesion and invasion ⁵⁹ . Phosphorylation of the myosin heavy chain at a conserved tyrosine residue was detected in response to Lm infection ⁶⁰ . Although phosphorylation of this tyrosine has been previously predicted in muscle myosin heavy chain ⁶¹ , its impact on myosin contractility is unknown. Myosin activity seems to protect plasma membrane integrity from LLO-induced damage and this leads to increased host survival in vivo in a zebrafish infection model ⁶² , although the underlying mechanism remains unclear.

Furthermore, formin and the actomyosin regulator Rho-associated kinase (ROCK) induce the internalization of Lm into endothelial cells ⁶³ . While in other cell types (such as epithelial and fibroblast) ROCK inhibits the entry of Lm, ROCK appears to favor bacterial adhesion to the cell surface of endothelia ⁶³ . It will be interesting to see if other regulators of actomyosin cortical tension cell–cell adhesions (for example 64) are involved in Lm infection.

Lm is known to alter the host endoplasmic reticulum (ER). Indeed, Lm induces ER stress and the unfolded protein response ⁶⁵ . The coat complex COPII, required for ER-to-Golgi trafficking ⁶⁶ , was recently found to restrict Lm cell-to-cell spread in polarized epithelial tissue culture ⁶⁷ . In addition, we discovered that Lm infection induces the expression of the small ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) in non-phagocytic cells, triggering an ISGylation of a number of ER and Golgi proteins and increasing cytokine secretion ⁶⁸ . Furthermore, studies have uncovered a novel role for Gp96 (glycoprotein of 96kDa), an ER resident protein chaperone. Lm infection was already known to trigger Gp96 recruitment from the ER to the plasma membrane, becoming exposed to the cell surface and co-localizing with surface bacteria ^{69, 70} . Recently, Gp96 was shown to be recruited to sites of LLO-induced blebbing along with myosin ⁶² , but the underlying mechanisms are unclear. It will be interesting to see if there are other strategies used by Lm to perturb trafficking between endomembrane components, especially in the context of different cell types.

Lm pathogenesis relies on the ability of the bacterium to traverse several physiological barriers, including the intestinal epithelium and the placenta, and survive in multiple cell types ⁷¹ .

Passage through the intestinal epithelial barrier is the first port of entry for Lm into the host. Interaction of the Lm surface protein InlA with E-Cadherin (E-cad), the host adherens junction epithelial cadherin is the key step in Lm intestinal invasion. Although E-Cad is localized to the basolateral membrane of vertebrate epithelial cells and would thus be generally inaccessible to Lm in the intestinal lumen, E-Cad is accessible at extruding cells at intestinal villi tips ⁷² and in mucus-secreting goblet cells ⁷³ . The interaction between InlA and human E-Cad is species specific ⁷⁴ . Thus, a knock-in transgenic mouse line bearing a point mutation in E-Cad that allows for the InlA–E-Cad interaction is used for oral infections with Lm ^{75, 76} , although many studies are still performed with non-transgenic mice ²¹ . InlA–E-Cad interaction triggers rapid transcytosis of Lm through goblet cells into the basal lamina propria ⁷³ . Recent work has shown that phosphoinositide 3-kinase (PI3-K) is constitutively active in the intestine, explaining why the Lm surface protein InlB, which is known to activate PI3-K, is not required for crossing the intestinal barrier ⁷⁷ . Interestingly, it was shown that Lm is mostly extracellular in the intestine of orally infected mice and that the intracellular pool is a minor but important fraction during infection ⁷⁸ . The majority of Lm in the gut was discovered to be associated with monocytes, but there is very poor intracellular growth ⁷⁹ in these cells.

Lm is one of the few pathogens capable of traversing the placental barrier. It requires both InlA and InlB ^{75, 80} . InlA-mediated invasion of Lm into the placenta requires InlB-dependent activation of PI3-K ⁷⁷ . Furthermore, a new Inl, InlP, has been discovered as an enhancer of placental invasion in both human placental explants and in vivo infection of guinea pigs and mice ⁸¹ , although the mechanisms through which InlP acts remain to be elucidated.