The abilities of bacterial pathogens to adapt to the environment within the host are essential to their virulence. Microorganisms have adapted to the iron limitation present in mammalian hosts by evolving diverse mechanisms for the assimilation of iron sufficient for growth. In addition, many bacterial pathogens have used the low concentration of iron present in the host as an important signal to enhance the expression of a wide variety of bacterial toxins and other virulence determinants. The molecular basis of coordinate regulation by iron has been most thoroughly studied in Escherichia coli. In this organism, coordinate regulation of gene expression by iron depends on the regulatory gene, fur. Regulation of gene expression by iron in a number of pathogenic organisms is coordinated by proteins homologous to the Fur protein of E. coli. Additional regulatory proteins may be superimposed on the Fur repressor to provide the fine-tuning necessary for the precise regulation of individual virulence genes in response to iron and other environmental signals. Studies of the mechanisms of regulation of iron acquisition systems and virulence determinants by iron should lead to a better understanding of the adaptive response of bacteria to the low-iron environment of the host and its importance in virulence.

We developed a multiplexed indirect immunofluorescent assay for antibodies to pneumococcal polysaccharides (PnPs) based on the Luminex multiple analyte profiling system (Luminex, Austin, TX). The assay simultaneously determines serum IgG concentrations to 14 PnPs serotypes: 1, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 12F; 14, 18C, 19F, and 23F. To assess the specificity of the multiplexed assay for each individual serotype, inhibition-of-binding studies were conducted using adult serum samples obtained after pneumococcal vaccination. Except for the closely related serotypes 9V and 9N, we demonstrated inhibition by homologous serotypes of more than 95% and inhibition by heterologous serotypes of less than 15% for all 14 PnPs serotypes. There was, however, high heterologous inhibition of 50% or greater with some serotypes. These cross-reacting antibodies could not be removed by preabsorption with pneumococcal C-polysaccharide but were removed by additional preabsorption with serotype 22F polysaccharide. The multiplexed Luminex assay showed good overall agreement with a well-established enzyme-linked immunosorbent assay that is currently recommended for evaluation of pneumococcal vaccine immunogenicity.

Many genes involved in the transport of iron by bacteria as well as in pathogenesis are regulated by the environmental concentration of iron, with increased expression under low-iron conditions. In Escherichia coli,

The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on the fur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae vibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. Vibrio vulnificus is a halophilic marine bacterium that has been associated with primary septicemia and serious wound infections (7,39,40,42). Primary septicemia is often acquired by eating raw oysters or shellfish, and wound infections are associated with exposure of wounds to seawater (30, 62). Primary septicemia is often associated with patients who have diseases predisposing them to iron overload, such as cirrhosis, hemochromatosis, and alcoholism, or who are immunocompromised (28).Iron is an essential element for the growth of most bacteria. In the mammalian host, most intracellular iron is found as heme, ferritin, hemoglobin, and hemosiderin. The concentration of available iron in the extracellular environment is extremely low because of the binding of iron to host high-affinity iron-binding proteins, such as transferrin and lactoferrin (3). Bacteria have evolved various mechanisms for the acquisition of iron from the host, including specific uptake of iron-chelating siderophores or the use of host iron compounds directly. Production of these iron uptake systems is repressed in the presence of iron by an iron-binding repressor protein called Fur (for ferric uptake regulation) (2).Iron appears to be particularly important in the pathogenesis of V. vulnificus infections. Stelma et al. (60) found that iron overload was a more significant risk factor for infection than impaired immune function. Virulent isolates were resistant to inactivation by serum complement, produced a phenolate (catechol) siderophore, had high titers of hemolysin, and utilized transferrin-bound iron. Avirulent isolates in that study were unable either to utilize transferrin-bound iron or to produce significant amounts of catechol siderophore. The results suggest that the phenolate (catechol) siderophore enables the virulent isolates to acquire iron from highly saturated transferrin. Morris et al. (41) also found a significant association between virulence and the utilization of transferrin as an iron source. The structure of the phenolate siderophore of V. vulnificus, named vulnibactin, has been characterized (47). We recently isolated a V. vulnificus mutant that was unable to produce catechol siderophores or to acquire iron from transferrin (34). This mutant showed reduced virulence in an infant mouse model.A V. vulnificus fur deletion mutant overexpresses at least two normally iron-regul...

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in immunocompromised individuals and patients who have hemochromatosis, cirrhosis, or alcoholism (3,41,42). Many of the V. vulnificus infections occur in patients with iron overload. Septicemia is often acquired by eating shellfish, and the mortality rates of patients with septicemia often exceed 50% (26).A number of factors have been proposed as possible virulence determinants for V. vulnificus, including an extracellular cytolysin (20,21,30), an elastolytic protease (27,28), siderophores (54), a phospholipase (61), the presence of a polysaccharide capsule (29,31,56,64,65), resistance to the bactericidal effects of sera (25,64,65), resistance to phagocytosis (25,29,65), and the ability to acquire iron from transferrin (44, 55, 56). Wright et al. directly correlated virulence of V. vulnificus with the availability of iron (63).

Objective: Previous studies suggest that gender affects the adaptive responses of the heart to some forms of cardiac overload. It is unknown whether gender influences left ventricular (LV) remodeling after myocardial infarction (MI). Methods: We performed transthoracic echocardiographic-Doppler examinations in age-matched male (n = 17) and female (n = 16) rats before, and 1 and 6 weeks after transmural MI or sham surgery. Results: Following large MI (male = 45 ± 1% LV circumference vs. female = 48 ± 4%, p = NS), both male and female rats developed progressive LV dilatation. Infarctions caused a similar degree of global and regional LV systolic dysfunction in males and females. Male rats had significant increases in the thickness of the noninfarcted posterior wall by 6 weeks after MI. However, posterior wall thickness did not change in the infarcted female rats. Average myocyte diameter in the noninfarcted region of the heart was also greater in male than female MI rats. The combination of increased cavity size with little change in wall thickness resulted in a greater decline in relative wall thickness in the female rats compared to the males. Male rats with MI showed progressively restricted LV diastolic filling as assessed by transmitral Doppler recordings. Female rats had less of an increase in the ratio of early to late transmitral velocities and less of an increase in the E wave deceleration rate after MI. Conclusions: Female rats showed a different pattern of LV remodeling than males with less of an increase in thickness of the noninfarcted portions of the left ventricle than males, but comparable LV cavity enlargement and systolic dysfunction. Despite similar infarct size, females developed less pronounced abnormalities of LV diastolic filling. We hypothesize that the gender-related differences in postinfarction LV remodeling may contribute to the different LV filling patterns, and might ultimately relate to differences in clinical outcome.

Iron concentration influences the expression of a number of genes involved in iron uptake and virulence in bacteria. In Escherichia coli, coordinate regulation of these genes by iron depends on the product of thefUr gene, which acts as an iron-responsive, DNA-binding repressor protein. Several genes in Vibrio cholerae are also repressed by iron; and afur gene, homologous to E. colifur, has been previously cloned from this organism. The present study was undertaken to define the roles of Fur and iron in regulating gene expression in V. chokrae.V. cholerae strains with a mutation in fir by virtue of suicide plasmid integration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentrations of iron; even in thefur mutants, however, residual two-to threefold regulation by iron persisted. Theifur mutant strains constructed by suicide plasmid integration required antibiotic selection to maintain the mutation. To analyze further the effect of Fur and iron on gene regulation in V. chokrae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V. cholerae fur mutant. This V. cholerae fur mutant grew significantly less well in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions. The V. cholerae fur mutant was unable to utilize a number of carbon sources including glycerol, acetate, succinate, lactate, and fumarate, that supported growth of the wild-type strain on minimal media. We utilized two-dimensional gel electrophoresis of whole-cell protein extracts from thefur mutant and wild-type strains following growth in conditions of either low or high concentrations of iron to identify proteins regulated by iron and/or Fur. Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent repressor. However, many other proteins were regulated in a different manner by iron and/or Fur. Seventeen proteins were negatively regulated by iron but independent of Fur, suggesting the presence of an additional iron-dependent repressor(s). Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain regardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism. There were 11 proteins that were induced rather than repressed by iron, in four different regulatory classes. Gene regulation in V. cholerae by Fur and iron is much more complex than previously thought and is reminiscent of the Lrp regulon in E. coli.Iron is an important element required by bacteria for growth and survival. Free iron is limited in the extracellular environment of the mammalian host (6), and bacteria respond to this iron limitation either by secreting low-molecular-weight siderophores to remove iron from host iron-binding proteins or by acquiring iron directly ...

Background. Previous studies have shown that global left ventricular function is depressed after myocardial infarction. However, little is known about the effects of myocardial infarction on contractility and the passive-elastic properties of residual myocardium.Methods and Results. We evaluated isometric function and passive myocardial stiffness in isolated, noninfarcted left ventricular papillary muscle from rats 6 weeks after sham operation or myocardial infarction. Maximal developed tension and peak rate of tension rise (+dT/dt) were significantly decreased in untreated rats with large myocardial infarction compared with controls (3.3+1.1 versus 43+0.6 g/mm2 and 49.5±f17.5 versus 72.5±+10.5 g/mm2/sec, respectively). Time to peak tension was prolonged (120±8 versus 102±4 msec) and myocardial stiffness was increased in untreated myocardial infarction rats compared with controls (35.2±4.9 versus 24.2±3.7). Rats with smaller myocardial infarctions differed from controls only with respect to a prolongation of time to peak tension. Papillary muscle myocyte cross-sectional area was increased by 44% (p<0.05), and myocardial hydroxyproline content was increased by 160% (p<0.05) in rats with large myocardial infarctions compared with controls. To determine whether treatment that improves left ventricular function after myocardial infarction also improves myocardial function, rats were treated with captopril beginning 3 weeks after myocardial infarction and continuing for 3 weeks. Treatment with captopril attenuated the prolongation in time to peak tension in the myocardial infarction rats; however, developed tension, +dT/dt, and muscle stiffness remained abnormal. Compared with untreated myocardial infarction rats, captopril-treated myocardial infarction rats had a 9% decrease in myocyte cross-sectional area (p=0.1) but a persistent increase in myocardial collagen content. In summary, large myocardial infarction in rats causes contractile dysfunction, increased stiffness, myocyte hypertrophy, and increased collagen content in the residual noninfarcted myocardium. Treatment with captopril alters the process of cardiac remodeling and hypertrophy and improves one parameter of contractility in noninfarcted myocardium; however, myocardial collagen content and myocardial stiffness remain abnormal.Conclusions. These findings suggest that angiotensin converting enzyme inhibition in the rat infarct model of heart failure improves global cardiac performance via combined effects on myocardial function and the peripheral circulation.