Isabel Martinez-Argudo

Translocation of enteropathogenic Escherichia coli across an in vitro M cell model is regulated by its type III secretion system

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that utilizes a type III secretion system (TTSS) to modulate diverse host cell processes including cytoskeletal dynamics, tight junction permeability and macrophage phagocytosis. Some EPEC strains exhibit selective tropism for the specialized follicle‐associated epithelium (FAE) overlying lymphoid follicles in the gut, which is a major site of uptake of inert particulates and pathogens, but do not translocate from the intestinal lumen in significant numbers. We have investigated the interaction of EPEC with FAE using an established in vitro model of the specialized FAE in which polarized enterocyte‐like Caco‐2 cells cocultured with the Raji B cell line undergo a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen‐transporting M cells found within FAE. Having confirmed that coculture with Raji B cells induces brush border reorganization and enhances particle transport across Caco‐2 cells, we investigated translocation of bacteria across the M cell model. While Salmonella translocation was markedly upregulated by Raji coculture, transport of wild‐type EPEC occurred at similarly low levels across both native Caco‐2 and Caco‐2/Raji‐cocultured layers. Translocation rates were markedly higher for EPEC strains lacking either functional TTSS or the effector protein EspF. These observations resemble previously reported data on the inhibition of macrophage phagocytosis by EPEC, which has also been reported to be dependent on TTSS and EspF. Furthermore, as with macrophage phagocytosis, enhanced translocation of a TTSS mutant was blocked by wortmannin, implicating inhibition of phosphatidyl inositol 3‐kinase‐mediated signalling in the regulation of M cell translocation by EPEC.

Salmonella translocates across an in vitro M cell model independently of SPI-1 and SPI-2

We have used an in vitro model of intestinal M cells to examine the mechanisms by which Salmonella enterica translocates across these specialized cells, which constitute a primary site of infection of the mammalian host. S. enterica can invade cultured cells by deploying a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI-1) to translocate effector proteins into the host cell cytoplasm that trigger cellular responses, including prominent cytoskeletal rearrangements. After Salmonella enters the host cell, a second TTSS encoded in SPI-2 modulates intracellular trafficking and enables the bacteria to replicate within a modified vacuolar compartment. Within the host intestine, specialized antigen-sampling M cells, which reside in the epithelium overlying lymphoid tissues in the gut, are a preferential site of Salmonella invasion. The mechanisms of infection of M cells remain poorly defined and it is not known whether either SPI-1 or SPI-2 is required for infection of these cells. To address these questions we have employed an in vitro M cell model involving co-culture of polarized Caco-2 intestinal epithelial cells with Raji B cells. S. enterica serovar Typhimurium translocated across Caco-2/Raji co-cultures to a much greater extent than they cross native Caco-2 cell monolayers. Salmonella translocation was greatly reduced by heat treatment or fixation, suggesting that processes distinct from the sampling of inert particles are the main determinants of bacterial translocation. Translocation across both mono-cultured and co-cultured Caco-2 cells was partially inhibited by treatment with the dynamin inhibitor dynasore, but resistant to EIPA, an inhibitor of macropinocytosis. There was no difference between the abilities of wild-type Salmonella Typhimurium and mutants lacking multiple SPI-1 effectors to translocate across the M cell model, although the SPI-1 effector mutants were somewhat attenuated for translocation across native Caco-2 layers. There was also no difference between wild-type and SPI-2 mutants in M cell translocation. Together these data suggest that that SPI-1 and SPI-2 are dispensable for rapid M cell translocation and that infection at these specialized epithelial sites involves distinctive mechanisms that are not reliably modelled using conventional cell culture infection models.

The extreme C terminus of Shigella flexneri IpaB is required for regulation of type III secretion, needle tip composition, and binding

ABSTRACT Type III secretion systems (T3SSs) are widely distributed virulence determinants of Gram-negative bacteria. They translocate bacterial proteins into host cells to manipulate them during infection. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region, and a hollow needle protruding from the bacterial surface. The distal tip of mature, quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. However, its mode of needle association is unknown. Here, we show that deletion of 3 or 9 residues at the C terminus of IpaB leads to fast constitutive secretion of late effectors, as observed in a ΔipaB strain. Like the ΔipaB mutant, mutants with C-terminal mutations also display hyperadhesion. However, unlike the ΔipaB mutant, they are still invasive and able to lyse the internalization vacuole with nearly wild-type efficiency. Finally, the mutant proteins show decreased association with needles and increased recruitment of IpaC. Taken together, these data support the notion that the state of the tip complex regulates secretion. We propose a model where the quiescent needle tip has an “off” conformation that turns “on” upon host cell contact. Our mutants may adopt a partially “on” conformation that activates secretion and is capable of recruiting some IpaC to insert pores into host cell membranes and allow invasion.

Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram‐negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaDs C‐terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.

Three‐dimensional electron microscopy reconstruction and cysteine‐mediated crosslinking provide a model of the type III secretion system needle tip complex

Type III secretion systems are found in many Gram‐negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the devices external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild‐type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three‐dimensional images of TCs at ∼20u2009Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease‐protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE+ and SopE− S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH–gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA–gfp reporter mirrored that of PprgH–gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression

We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH::gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR, should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

ABSTRACT The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127 is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127 has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of different espF alleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. The espF gene from E. coli serotype O157 (espF O157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF O127 and espF O26. In contrast, espF O157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

Isolation of Salmonella Mutants Resistant to the Inhibitory Effect of Salicylidene acylhydrazides on Flagella-Mediated Motility

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB (P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors molecular target(s) using motility assays in the ΔfliH-fliI/flhB (P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18s phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.

Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.