RNA-Seq analysis of the T3SA regulon in Shigella flexneri reveals two new chromosomal genes upregulated in the on-state.

Shigella spp. are enterobacteria that invade human colonic mucosal cells using their Type Three Secretion Apparatus (T3SA). Shigella spp. possess a large plasmid that encodes most of its virulence factors and has been the focus of seminal work that defined the T3SA regulon. Thus, a global assessment of the transcriptional response regulated by the T3SA has been lacking. Herein we used RNA-Seq to identify genes that are differentially expressed when the T3SA is active (on-state) versus inactive (off-state). The quality of the RNA-Seq dataset was validated by its correlation with a prior microarray study. Using novel insights about the expression of non-coding regions, bioinformatic tools and experimentations, we demonstrated the existence of six operons and evidence that ipaH2.5 is a pseudogene. In addition, 86 chromosomal genes were downregulated in the on-state including several non-coding transcripts corresponding to short antisense RNA embedded in the 16S and 23S RNA genes, and 40 coding transcripts, whose cognate proteins were highly connected at the genetic and biochemical levels. Finally, we identified two novel chromosomal genes dubbed gem1 and gem3, which were upregulated in the on-state similarly to genes belonging to the T3SA regulon. The latter findings were validated on biological triplicates by droplet digital PCR. To our knowledge gem1 and gem3 are the first chromosomal members of the T3SA regulon that have no homologs on the plasmid. Our approach provides a path to optimizing RNA-Seq studies in case of bacterial models that had previously been the subject of medium to large scale studies.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally.

Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed.

Tracking bacterial pathogens with genetically-encoded reporters.

During the infectious process, bacterial pathogens are subject to changes in environmental conditions such as nutrient availability, immune response challenges, bacterial density and physical contacts with targeted host cells. These conditions occur in the colonized organs, in diverse regions within infected tissues or even at the subcellular level for intracellular pathogens. Integration of environmental cues leads to measurable biological responses in the bacterium required for adaptation. Recent progress in technology enabled the study of bacterial adaptation in situ using genetically encoded reporters that allow single cell analysis or whole body imaging based on fluorescent proteins, alternative fluorescent assays or luciferases. This review presents a historical perspective and technical details on the methods used to develop transcriptional reporters, protein-protein interaction assays and secretion detection assays to study pathogenic bacteria adaptation in situ. Finally, studies published in the last 5 years on gram positive and gram negative bacterial adaptation to the host during infection are discussed. However, the methods described here could easily be extended to study complex microbial communities within host tissue and in the environment.

Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase.

Biochemical 'pathways' are systems of dynamically assembling and disassembling protein complexes, and thus, much of modern biological research is concerned with how, when and where proteins interact with other proteins involved in biochemical processes. The demand for simple approaches to study protein-protein interactions, particularly on a large scale, has grown recently with the progress in genome projects, as the association of unknown with known gene products provides one crucial way of establishing the function of a gene. It was with this challenge in mind that our laboratory developed a simple survival protein-fragment complementation assay (PCA) based on the enzyme dihydrofolate reductase (DHFR). In the DHFR PCA strategy, two proteins of interest are fused to complementary fragments of DHFR. If the proteins of interest interact physically, the DHFR complementary fragments are brought together and fold into the native structure of the enzyme, reconstituting its activity, detectable by the survival of cells expressing the fusion proteins and growth in selective medium. Using the protocol described here, the survival selection can be completed in one to several days, depending on the cell type.

The transition state of the ras binding domain of Raf is structurally polarized based on Phi-values but is energetically diffuse.

The ras binding domain (RBD) of the Ser/Thr kinase c-Raf/Raf-1 spans 78 residues and adopts a structure characteristic of the beta-grasp ubiquitin-like topology. Recently, the primary sequence of Raf RBD has been nearly exhaustively mutated experimentally by insertion of stretches of degenerate codons, which revealed sequence conservation and hydrophobic core organization similar to that found in an alignment of beta-grasp ubiquitin-like proteins. These results now allow us to examine the relationship between sequence conservation and the folding process, particularly viewed through the analysis of transition state (TS) structure. Specifically, we present herein a protein engineering study combining classic truncation (Ala/Gly) and atypical mutants to predict folding TS ensemble properties. Based on classical Phi-value analysis, Raf RBD TS structure is particularly polarized around the N-terminal beta-hairpin. However, all residues constituting the inner layer of the hydrophobic core are involved in TS stabilization, although they are clearly found in a less native-like environment. The TS structure can also be probed by a direct measure of its destabilization upon mutation, DeltaDeltaG(U-++). Viewed through this analysis, Raf RBD TS is a more diffuse structure, in which all residues of the hydrophobic core including beta-strands 1, 2, 3 and 5 and the major alpha-helix play similar roles in TS stabilization. In addition, Phi-values and DeltaDeltaG(U-++) reveal striking similarities in the TS of Raf RBD and ubiquitin, a structural analogue displaying insignificant sequence identity (<12%). However, ubiquitin TS appears more denatured-like and polarized around the N-terminal beta-hairpin. We suggest that analysis of Phi-values should also consider the direct impact of mutations on differences in free energy between the unfolded and TS (DeltaDeltaG(U-++)) to ensure that the description of TS properties is accurate. Finally, the impact of these findings on the modeling of protein folding is discussed.

Massive sequence perturbation of the Raf ras binding domain reveals relationships between sequence conservation, secondary structure propensity, hydrophobic core organization and stability.

The contributions of specific residues to the delicate balance between function, stability and folding rates could be determined, in part by [corrected] comparing the sequences of structures having identical folds, but insignificant sequence homology. Recently, we have devised an experimental strategy to thoroughly explore residue substitutions consistent with a specific class of structure. Using this approach, the amino acids tolerated at virtually all residues of the c-Raf/Raf1 ras binding domain (Raf RBD), an exemplar of the common beta-grasp ubiquitin-like topology, were obtained and used to define the sequence determinants of this fold. Herein, we present analyses suggesting that more subtle sequence selection pressure, including propensity for secondary structure, the hydrophobic core organization and charge distribution are imposed on the Raf RBD sequence. Secondly, using the Gibbs free energies (DeltaG(F-U)) obtained for 51 mutants of Raf RBD, we demonstrate a strong correlation between amino acid conservation and the destabilization induced by truncating mutants. In addition, four mutants are shown to significantly stabilize Raf RBD native structure. Two of these mutations, including the well-studied R89L, are known to severely compromise binding affinity for ras. Another stabilized mutant consisted of a deletion of amino acid residues E104-K106. This deletion naturally occurs in the homologues a-Raf and b-Raf and could indicate functional divergence. Finally, the combination of mutations affecting five of 78 residues of Raf RBD results in stabilization of the structure by approximately 12 kJ mol(-1) (DeltaG(F-U) is -22 and -34 kJ mol(-1) for wt and mutant, respectively). The sequence perturbation approach combined with sequence/structure analysis of the ubiquitin-like fold provide a basis for the identification of sequence-specific requirements for function, stability and folding rate of the Raf RBD and structural analogues, highlighting the utility of conservation profiles as predictive tools of structural organization.

Massive sequence perturbation of a small protein.

Most protein topologies rarely occur in nature, thus limiting our ability to extract sequence information that could be used to predict structure, function, and evolutionary constraints on protein folds. In principle, the sequence diversity explored by a given protein topology could be expanded by introducing sequence perturbations and selecting variant proteins that fold correctly. However, our capacity to explore sequence space is intrinsically limited by the enormous number of sequences generated from the 20 amino acids and the limited number of variants likely to fold. Here we sought to test whether the sequence space for naturally existing proteins can be explored by simple, sequential degeneration of a complete set of short sequence segments of a model protein, without long-range covariation. Using the Raf ras binding domain as a model of a small protein capable of autonomous folding, we degenerated 72 of 76 positions of the primary structure for the 20 amino acids in segments of four to seven residues defined by secondary structure and selected the folded species for interaction with h-ras by using an in vivo survival-selection assay. The methodology presented allowed for rigorous statistical analysis and comparison of sequence diversity. The ensemble of sequence variants of Raf ras binding domain obtained have recaptured the diversity observed for the ubiquitin-roll topology. A signature sequence for this fold and the implication of this strategy to protein design and structure prediction are discussed.

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments.

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.