Fibrin and fibrinolysis in infection and host defense.

Bacterial pathogens have frequently evolved and maintained the capacity to engage and/or activate hemostatic system components of their vertebrate hosts. Recent studies of mice with selected alterations in host plasminogen and other hemostatic factors have begun to reveal a seminal role of bacterial plasminogen activators and fibrin clearance in microbial pathogenesis. Bacterial pathogens appear to exploit host plasmin-mediated proteolysis to both support microbial dissemination and evade innate immune surveillance systems. The contribution of bacterial plasminogen activation to the evasion of the inflammatory response is particularly conspicuous with the plague agent, Yersinia pestis. Infection of control mice with wild-type Y. pestis leads to the formation of widespread foci containing massive numbers of free bacteria with little inflammatory cell infiltrate, whereas the loss of either the bacterial plasminogen activator, Pla, or the elimination of host plasminogen results in the accumulation of robust inflammatory cell infiltrates at sites of infection and greatly improved survival. Interestingly, fibrin(ogen) deficiency undermines the local inflammatory response observed with Pla-deficient Y. pestis and effectively eliminates the survival benefits posed by the elimination of either host plasminogen or bacterial Pla. These studies, and complementary studies with other human pathogens, illustrate that plasminogen and fibrinogen are extremely effective modifiers of the inflammatory response in vivo and critical determinants of bacterial virulence and host defense. Detailed studies of the inflammatory response in mice with genetically-imposed modifications in coagulation and fibrinolytic factors underscore the regulatory crosstalk between the hemostatic and immune systems.

RNA expression patterns change dramatically in human neutrophils exposed to bacteria.

A comprehensive study of changes in messenger RNA (mRNA) levels in human neutrophils following exposure to bacteria is described. Within 2 hours there are dramatic changes in the levels of several hundred mRNAs including those for a variety of cytokines, receptors, apoptosis-regulating products, and membrane trafficking regulators. In addition, there are a large number of up-regulated mRNAs that appear to represent a common core of activation response genes that have been identified as early-response products to a variety of stimuli in a number of other cell types. The activation response of neutrophils to nonpathogenic bacteria is greatly altered by exposure to Yersinia pestis, which may be a major factor contributing to the virulence and rapid progression of plague. Several gene clusters were created based on the patterns of gene induction caused by different bacteria. These clusters were consistent with those found by a principal components analysis. A number of the changes could be interpreted in terms of neutrophil physiology and the known functions of the genes. These findings indicate that active regulation of gene expression plays a major role in the neutrophil contribution to the cellular inflammatory response. Interruption of these changes by pathogens, such as Y pestis, could be responsible, at least in part, for the failure to contain infections by highly virulent organisms.

Role of the pleiotropic effects of plasminogen deficiency in infection experiments with plasminogen-deficient mice.

Plasminogen-deficient mice hold great promise as tools for analyzing the contribution of plasminogen activators produced by infectious agents to pathogenesis. However, the pathology caused by congenital plasminogen deficiency complicates the interpretation of infection experiments conducted with these animals. This pathology, the most prominent features of which are poor weight gain, wasting after about 60 days of age, and shortened lifespan, results from the inability of the mice to clear small fibrin thrombi. This article describes strategies for distinguishing the contribution of this pathology from the direct effects of depriving infectious agents of plasminogen. These strategies depend on the use of mouse genotypes in which the correlation of plasminogen deficiency with fibrin-dependent pathology is broken. Mice with plasminogen activator deficiencies are unable to generate plasmin and develop pathologies identical to those seen in plasminogen-deficient mice. However, unlike plasminogen-deficient mice, they do make plasminogen available to the infectious agent. Fibrinogen-deficient mice also deficient for plasminogen do not develop the pathology typical of plasminogen deficiency. These mice allow examination of plasminogen deficiency in the absence of fibrin-dependent pathology. Use of fibrinogen-deficient mice is complicated by the possibility that fibrin may be the key substrate of plasmin generated by the infectious agent.

Proteases and bacterial virulence: a view from the trenches.

Many species of pathogenic bacteria produce cell-surface or secreted proteases. These enzymes have high potential to enhance bacterial pathogenesis through degradation of critical host proteins and by mimicking the activity of host regulatory proteases that control important zymogen systems. Although many bacterial proteases have been implicated in virulence, there is currently no system in which both rigorous demonstration of virulence enhancement in vivo and convincing identification of the important substrate molecules has been achieved. The difficulties inherent in addressing these issues is discussed, and several interesting systems under active investigation briefly described. The potential of extracellular protease as targets for drug development is also considered.

Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.

A surface protease and the invasive character of plague.

A 9.5-kilobase plasmid of Yersinia pestis, the causative agent of plague, is required for high virulence when mice are inoculated with the bacterium by subcutaneous injection. Inactivation of the plasmid gene pla, which encodes a surface protease, increased the median lethal dose of the bacteria for mice by a millionfold. Moreover, cloned pla was sufficient to restore segregants lacking the entire pla-bearing plasmid to full virulence. Both pla+ strains injected subcutaneously and pla- mutants injected intravenously reached high titers in liver and spleen of infected mice, whereas pla- mutants injected subcutaneously failed to do so even though they establish a sustained local infection at the injection site. More inflammatory cells accumulated in lesions caused by the pla- mutants than in lesions produced by the pla+ parent. The Pla protease was shown to be a plasminogen activator with unusual kinetic properties. It can also cleave complement C3 at a specific site.

Temperature sensing in Yersinia pestis: regulation of yopE transcription by lcrF.

In Escherichia coli, a yopE::lacZ fusion was found to be regulated by temperature in the presence of the cloned BamHI G fragment of Yersinia pestis plasmid pCD1, which contains the lcrF locus. Increasing the copy number of lcrF relative to that of the yopE reporter had a negligible effect on the induction ratio (26 versus 37 degrees C) but caused large reductions in the absolute levels of yopE transcription. We localized the lcrF gene by monitoring the induction phenotype of BamHI G deletion derivatives. Sequencing revealed an open reading frame capable of encoding a protein of 30.8 kDa. A protein product of this size was detected in a T7 expression system, and LcrF-dependent yopE-specific DNA binding activity was observed. As expected, LcrF exhibited 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcriptional regulatory proteins. These proteins could be divided into two classes according to function: those regulating operons involved in catabolism of carbon and energy sources and those involved in regulating virulence genes. lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. A portion of LcrF was found associated with the membrane fraction in E. coli; however, pulse-chase experiments indicated that this result is an artifact of fractionation.

Triton's Global Heat Budget.

Internal heat flow from radioactive decay in Triton's interior along with absorbed thermal energy from Neptune total 5 to 20 percent of the insolation absorbed by Triton, thus comprising a significant fraction of Triton's surface energy balance. These additional energy inputs can raise Triton's surface temperature between approximately 0.5 and 1.5 K above that possible with absorbed sunlight alone, resulting in an increase of about a factor of approximately 1.5 to 2.5 in Triton's basal atmospheric pressure. If Triton's internal heat flow is concentrated in some areas, as is likely, local effects such as enhanced sublimation with subsequent modification of albedo could be quite large. Furthermore, indications of recent global albedo change on Triton suggest that Triton's surface temperature and pressure may not now be in steady state, further suggesting that atmospheric pressure on Triton was as much as ten times higher in the recent past.

Nucleotide sequence of the plasminogen activator gene of Yersinia pestis: relationship to ompT of Escherichia coli and gene E of Salmonella typhimurium.

We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene.

Genetic analysis of the 9.5-kilobase virulence plasmid of Yersinia pestis.

The 9.5-kilobase plasmid of Yersinia pestis determines plasminogen activator, coagulase, pesticin, and pesticin immunity activities. We have mapped and cloned the loci encoding these activities and demonstrated that both plasminogen activator and coagulase were determined by the same gene, designated pla. The primary translation product of this gene (38 kilodaltons [kDa]) was processed in two sequential steps to produce peptides of 37 and 35 kDa. The first step in this processing occurred rapidly and probably cotranslationally and was blocked when protein export was inhibited. The second step was much slower and resulted in the presence of both the 37- and 35-kDa species in significant quantities. We also showed that the plasmid had a polA-dependent replicon and identified the region that contained its origin of replication and incompatibility functions.

Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins.

The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid.

Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes).

The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae. Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E. coli to eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.

Genetic mapping of novel virulence determinants of Salmonella typhimurium to the region between trpD and supD.

Salmonella typhimurium strains are known to vary greatly in their virulence for mice. A few of the genes affecting their virulence have been described. In this report we have localized at least two genes that affect the ability of S. typhimurium to grow in BALB/cByJ to a 6 unit section of the salmonella chromosome which does not contain any previously described virulence determinants. The genetic mapping was done by interrupted matings using Hfr strains made in a virulent LT2 strain. The Hfr strains were constructed by inserted the plasmid F'(TS)114 lac+ Tn::10 into the LT2 chromosome at specific sites through homologous recombination with chromosomal Tn10s. Short interrupted matings to an avirulent LT2 strain in either direction through the portion of the chromosome from trpD at 34 units to supD at 40 units resulted in transconjugants which were fully virulent. Since we also found several transconjugants with intermediate virulence it appears that more than one virulence gene may exist in this area of the chromosome. The mechanisms of action of these genes are not known.

Plasmid-determined cytotoxicity in Yersinia pestis and Yersinia pseudotuberculosis.

Yersinia pestis KIM5 was found to be cytotoxic for the IC21 and P388D1 mouse macrophage cell lines, as well as for resident peritoneal macrophages from C57BL/6 mice. Affected cells phagocytosed KIM5 inefficiently, became spherical, detached readily from culture dishes, and retained 51Cr poorly. The cytotoxic effect was dependent on the presence of the 75-kilobase plasmid pCD1. Because this plasmid also encodes the low calcium response (LCR), three Mu d1 insertion mutants previously shown to be LCR- and of reduced virulence in mice were examined for cytotoxicity; all were found to be atoxic. The insertions in these mutants lie within three distinct LCR loci (lcrB, C, and D). Like LCR, cytotoxicity was expressed only at 37 degrees C. Unlike LCR, it was not influenced by Ca2+ concentration, indicating that the V and W antigens are probably not involved. Yersinia pseudotuberculosis was found to have a similar plasmid-dependent cytotoxicity. Thus, biological activity observed as cytotoxicity in vitro may well be a common feature contributing to virulence of the yersiniae.

The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit.

The role of type 1 fimbriae in the mannose-sensitive attachment of Escherichia coli to eucaryotic cells was investigated by deletion mutation analysis of a recombinant plasmid, pSH2, carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. A mutant, pUT2002, containing a deletion remote from the structural gene encoding the 17-kilodalton subunit protein of type 1 fimbriae failed to agglutinate guinea pig erythrocytes even though the bacteria expressed fimbriae morphologically and antigenically indistinguishable from those produced by the intact recombinant plasmid. Fimbriae isolated from pUT2002 failed to agglutinate guinea pig erythrocytes, but reacted with a monoclonal antibody specific for quaternary structural determinants of type 1 fimbriae. Moreover, the dissociated fimbrial subunits from this mutant were indistinguishable from normal fimbriae by their migration during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, by their reactivity with a monoclonal antibody directed against a subunit-specific epitope, and in enzyme-linked immunosorbent assays with monospecific antisera. These results indicate that the adhesive functions in type 1 fimbriae are dependent on a factor(s) encoded by a gene other than those required for synthesis, assembly, and expression of the structural 17-kilodalton subunit.

Temperature-controlled plasmid regulon associated with low calcium response in Yersinia pestis.

Both the low calcium response and virulence in Yersinia pestis strain KIM5 are mediated by genes located on the 75.4-kilobase plasmid pCD1. The results presented here demonstrate the existence of two new genetic loci of pCD1 whose expression is regulated in response to temperature. Levels of transcription in the trtA and trtB loci were elevated 12- to 16-fold at 37 degrees C compared with levels of transcription at 30 degrees C. In addition, the absolute levels of transcription were the highest that have been reported for genes on pCD1. Mutations in trtB also abolished production of the V antigen. Thermal induction at these loci was dramatically reduced in strains harboring a Tn5 insertion in the lcrF locus of pCD1. lcrF lies 33 and 13 kilobases from the trtA and trtB loci, respectively. Thus, lcrF is a positive regulatory gene responsible for temperature-induced transcription of genes required for the low calcium response.

The DNA sequence recognized by the EcoDXX1 restriction endonuclease.

EcoDXX1 is a type-I restriction enzyme coded for by the plasmid pDXX1. Like other type-I restriction endonucleases, the enzyme catalyses the modification of susceptible DNA. We have determined the DNA sequence recognised by EcoDXX1 to be: 5'TCANNNNNNNATTC-3' 3'-AGTNNNNNNNTAAG-5' where N can be any nucleotide. This sequence has an overall structure very similar to previously determined type-I sequences.

Identification and preliminary characterization of external membrane-bound nuclease activities in Mycoplasma pulmonis.

Mycoplasma pulmonis has substantial DNase activity exposed on the cell surface. At least part of this activity is attributable to an endonuclease. The activity is destroyed at 56 degrees C and inhibited by either 5 mM EDTA or 10 mM zinc chloride. It can also be eliminated by treatment of intact organisms with trypsin and is regenerated by incubation of the treated organisms in a medium that supports protein synthesis. DNase exposed at the cell surface constitutes 20% of the total DNase activity present in M. pulmonis extracts.

Isolation and characterization of Ca2+-blind mutants of Yersinia pestis.

The plasmid pCD1 is required for expression of the low-calcium response (LCR), virulence, and production of V antigen in Yersinia pestis KIM. Five independent mutants constitutive for the LCR at 37 degrees C (Lcrc) were obtained through ethyl methanesulfonate mutagenesis followed by ampicillin enrichment. A sixth, spontaneous mutant was obtained directly through ampicillin enrichment. These mutants failed to grow at 37 degrees C regardless of calcium concentration and produced V antigen constitutively at this temperature. All six mutations were located on pCD1. One mutation was mapped to a 1-kilobase region of lcrA. Based on complementation mapping of this mutation, the lcrA locus was divided into two new loci, lcrD and lcrE. This mutation, lcrE1, did not alter the transcription of other genes in the LCR region and was cis-recessive to lcr mutations. Several lower-molecular-weight outer membrane proteins which were observed in the parent strain grown at 37 degrees C in the presence of 2.5 mM calcium were reduced in quantity or absent from the mutant strain.