Yersinia enterocolitica TyeA, an intracellular regulator of the type III machinery, is required for specific targeting of YopE, YopH, YopM, and YopN into the cytosol of eukaryotic cells

Pathogenic Yersinia species employ type III machines to target effector Yops into the cytosol of eukaryotic cells. Yersinia tyeA mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907-1918, 1998). We have examined the injection of Yop proteins by tyeA mutant yersiniae with the digitonin fractionation technique. We find that tyeA mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.     

TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expression and is required for polarized translocation of Yop effectors

Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.     

Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis yopN,tyeA, sycN,yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and phosphospecific antibodies

Yersinia pestis, the causative agent of plague, exports a set of virulence proteins called Yops upon contact with eukaryotic cells. A subset of these Yops is translocated directly into the cytosol of host cells. In this study, a novel protein tag-based reporter system is used to measure the translocation of Yops into cultured eukaryotic cells. The reporter system uses a small bipartite phosphorylatable peptide tag, termed the Elk tag. Translocation of an Elk-tagged protein into eukaryotic cells results in host cell protein kinase-dependent phosphorylation of the tag at a specific serine residue, which can subsequently be detected with phosphospecific antibodies. The YopN, TyeA, SycN, YscB and LcrG proteins function to prevent Yop secretion before host cell contact. The role of these proteins was investigated in the translocation of Elk-tagged YopE (YopE129-Elk) and YopN (YopN293-Elk) into HeLa cells. Y. pestis yopN, tyeA, sycN and yscB deletion mutants showed reduced levels of YopE129-Elk phosphorylation compared with the parent strain, indicating that these mutants translocate reduced amounts of YopE. We also demonstrate that YopN293-Elk is translocated into HeLa cells and that this process is more efficient in a Yersinia yop polymutant strain lacking the six translocated effector Yops. Y. pestis sycN and yscB mutants translocated reduced amounts of YopN293-Elk; however, tyeA and lcrG mutants translocated higher amounts of YopN293-Elk compared with the parent strain. These data suggest that TyeA and LcrG function to suppress the secretion of YopN before host cell contact, whereas SycN and YscB facilitate YopN secretion and subsequent translocation.     

Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: one-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone

Pathogenic Yersiniae adhere to and kill macrophages by targeting some of their Yop proteins into the eukaryotic cytosol. There is debate about whether YopE targeting proceeds as a direct translocation of polypeptide between cells or in two distinct steps, each requiring specific signals for YopE secretion across the bacterial envelope and for translocation into the eukaryotic cytosol. Here, we used the selective solubilization of the eukaryotic plasma membrane with digitonin to measure Yop targeting during Yersinia infections of HeLa cells. YopE, YopH, YopM and YopN were found in the eukaryotic cytosol but not in the extracellular medium. When bound to SycE chaperone in the Yersinia cytoplasm, YopE residues 1–100 are necessary and sufficient for the targeting of hybrid neomycin phosphotransferase. Electron microscopic analysis failed to detect an extracellular intermediate of YopE targeting, suggesting a one-step translocation mechanism.     

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.     

Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y.enterocolitica is internalized inside the cytosol of PU5-1.8 macrophages by the YopB, D, N delivery apparatus.

The Yersinia Yop virulon is an anti-host system made up of four elements: (i) a type III secretion system called Ysc; (ii) a system designed to deliver bacterial proteins into eukaryotic target cells (YopB, YopD); (iii) a control element (YopN); and (iv) a set of intracellularly delivered proteins designed to disarm these cells or disrupt their communications (YopE, YopH and possibly others). YopM, another Yop protein, binds thrombin and is thus presumed to act as an extracellular effector. Here, we analyzed YopM from Y.enterocolitica and we wondered whether it could also be delivered inside eukaryotic cells. To answer this question we applied the Yop-Cya reporter strategy. Hybrids made of 141 or 100 N-terminal residues of YopM fused to Cya were delivered inside PU5-1.8 macrophages by recombinant Y.enterocolitica strains. YopB and YopD were required as translocators. Leakage of the reporters into the macrophage culture supernatant during the bacterial infection increased strongly when YopN was missing, showing that YopN is involved in the control of delivery of YopM inside eukaryotic cells. YopN itself was not delivered into the macrophages. In conclusion, YopM is translocated inside the eukaryotic cells and its physiopathological role should be revised or completed.     

The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.     

Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica

During infection, Yersinia enterocolitica exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 microM (low-calcium response). Yersiniae lacking yopN (lcrE), yscB, sycN, or tyeA do not inactivate the type III pathway even when the concentration of calcium is above 100 microM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of yopN gene sequences to the 5' end of the npt reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of yopN. Expression of plasmid-borne yopN, but not of yopN(1-294)-npt, complements the calcium-blind phenotype of yopN mutants. Surprisingly, yopN mutants respond to environmental changes in calcium concentration and secrete YopN(1-294)-Npt in the absence but not in the presence of calcium. tyeA is required for the low-calcium regulation of YopN(1-294)-Npt secretion, whereas sycN and yscB mutants fail to secrete YopN(1-294)-Npt in the presence of calcium. Experiments with yopN-npt fusions identified two other signals that regulate the secretion of YopN. yopN codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of yscB and sycN. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional tyeA. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.     

A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis

Human pathogenic Yersinia resist host defences, in part through the expression and delivery of a set of plasmid-encoded virulence proteins termed Yops. A number of these Yops are exported from the bacteria directly into the cytoplasm of their eukaryotic host's cells upon contact with these cells. The secreted YopN protein (also known as LcrE) is required to block Yop secretion in the presence of calcium in vitro or before contact with a eukaryotic cell in vivo. In this study, we characterize the role of the tyeA, sycN and yscB gene products in the regulation of Yop secretion in Yersinia pestis. Mutants specifically defective in the expression of TyeA, SycN or YscB were no longer able to block Yop secretion in the presence of calcium. In addition, the secretion of YopN was specifically reduced in both the sycN and the yscB deletion mutants. Protein cross-linking and immunoprecipitation studies in conjunction with yeast two-hybrid analyses showed that SycN and YscB interact with one another to form a SycN/YscB complex. Yeast three-hybrid analyses demonstrated that the SycN/YscB complex, but not SycN or YscB alone, specifically associates with YopN. SycN and YscB share amino acid sequence similarity and structural similarities with the specific Yop chaperones SycE and SycH. Together, these results indicate that a complex composed of SycN and YscB functions as a specific chaperone for YopN in Y. pestis.     

YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5 kDa). Its secretion required an intact Ysc apparatus and SycT (15.0 kDa, pI 4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia , producing a hybrid YopT–adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (ΔHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells.     

The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes

Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type III secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N-terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc-binding domain in YopE (residues 15–50) and YopH (residues 20–70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes.     

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.     

Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosis

Type III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild-type or Yop-Y. pseudotuberculosis strains were assayed for interleukin (IL)-8 production. HeLa cells infected with a YopEHJ- triple mutant released significantly more IL-8 than HeLa cells infected with isogenic wild-type, YopE-, YopH- or YopJ- bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL-8 production. IL-8 production required YopB, but did not require YopD, pore formation or invasin-mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen-activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ- mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells.     

A program of Yersinia enterocolitica type III secretion reactions is activated by specific signals

Successful establishment of Yersinia infections requires the type III machinery, a protein transporter that injects virulence factors (Yops) into macrophages. It is reported here that the Yersinia type III pathway responds to environmental signals by transporting proteins to distinct locations. Yersinia enterocolitica cells sense an increase in extracellular amino acids (glutamate, glutamine, aspartate, and asparagine) that results in the activation of the type III pathway. Another signal, provided by serum proteins such as albumin, triggers the secretion of YopD into the extracellular medium. The third signal, a decrease in calcium concentration, appears to be provided by host cells and causes Y. enterocolitica to transport YopE and presumably other virulence factors across the eukaryotic plasma membrane. Mutations in several genes encoding regulatory molecules (lcrG, lcrH, tyeA, yopD, yopN, yscM1, and yscM2) bypass the signal requirement of the type III pathway. Together these results suggest that yersiniae may have evolved distinct secretion reactions in response to environmental signals.     

Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE

A type III secretion-translocation system allows Yersinia adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca(2+)-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant Yersinia devoid of most of the Yop effectors (delta HOPEM and delta THE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE(15)-Cya as efficiently as YopE(130)-Cya. They were also able to deliver YopE(delta 17-77). SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type Yersinia suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca(2+) chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.     

A novel protein, LcrQ, involved in the low-calcium response of Yersinia pseudotuberculosis shows extensive homology to YopH.

The plasmid-encoded yop genes of pathogenic yersiniae are regulated by the environmental stimuli calcium and temperature. A novel protein, LcrQ, which exhibits a key function in the negative calcium-controlled pathway, was identified. DNA sequence analysis revealed that LcrQ has a molecular mass of 12,412 daltons and its isoelectric point is 6.51. Overexpression of LcrQ in trans in wild-type Yersinia pseudotuberculosis YPIII(pIB102) changed the phenotype from calcium dependence to calcium independence and inhibited Yop expression. LcrQ is expressed from a monocistronic operon. Trans overexpression of LcrQ in yopN and lcrH mutants affected the phenotype of the yopN mutant (temperature sensitive to calcium independence) but not that of the lcrH mutant (temperature sensitive), suggesting that LcrQ acts between YopN and LcrH in the calcium-regulated pathway. An lcrQ mutant was found to be temperature sensitive for growth and showed derepressed Yop expression at 37 degrees C in the presence of calcium in the growth medium. During these culture conditions, the lcrQ mutant secreted only LcrV and YopD into the culture supernatant. Removal of Ca2+ from the growth medium resulted in a Yop expression pattern of the mutant that was identical to that of the wild-type strain. The LcrQ protein was recovered from the culture supernatant. LcrQ shows 42% identity to the first 128 amino acids of the YopH virulence protein.     

Secretion of hybrid proteins by the Yersinia Yop export system.

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.