Capsule of Streptococcus pyogenes is essential for delayed death of mice in a model of streptococcal toxic shock syndrome

We have previously reported a mouse model of severe group A streptococcal infection (Microbiol. Immunol. 45: 777-786, 2001). When we injected Streptococcus pyogenes strains intramuscularly, the mice suffered from acute phase of infection for a few days but recovered from the illness and gained body weight. These mice, however, began to die after 3 weeks of infection, which we called 'delayed death.' Bacterial strains isolated from organs of the dead mice showed thick capsules. We, therefore, constructed a hyaluronic acid capsule gene, hasA, knockout mutant by homologous recombination and the effect of capsule on the death was observed. hasA knockout strain did not cause delayed death, though it caused acute death at high doses of infection. According to this result, the capsule is a critical pathogenic factor for causing the delayed death in our mouse model.     

Quantifying dose-, strain-, and tissue-specific kinetics of parainfluenza virus infection

Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.     

Deciphering the role of the capsule of Klebsiella pneumoniae during pathogenesis: A cautionary tale

Extracellular capsule polysaccharides increase the cellular fitness under abiotic stresses and during competition with other bacteria. They are best-known for their role in virulence, particularly in human hosts. Specifically, capsules facilitate tissue invasion by enhancing bacterial evasion from phagocytosis and protect cells from biocidal molecules. Klebsiella pneumoniae is a worrisome nosocomial pathogen with few known virulence factors, but the most important one is its capsule. In this issue, Tan et al. assess the fitness advantage of the capsule by competing a wild-type strain against four different mutants where capsule production is interrupted at different stages of the biosynthetic pathway. Strikingly, not all mutants provide a fitness advantage. They suggest that some mutants have secondary defects altering virulence-associated phenotypes and blurring the role of the capsule in pathogenesis. This study indicates that the K1 capsule in K. pneumoniae is not required for gut colonization but that it is critical for bloodstream dissemination to other organs. These results contribute to clarify the contradictory literature on the role of the Klebsiella capsule during infection. Finally, the varying fitness effects of different capsule mutations observed for K. pneumoniae K1 might apply also to other capsulated diderm bacteria that are facultative or emerging pathogens.     

Immunomodulatory effects of Streptococcus suis capsule type on human dendritic cell responses, phagocytosis and intracellular survival

Streptococcus suis is a major porcine pathogen of significant commercial importance worldwide and an emerging zoonotic pathogen of humans. Given the important sentinel role of mucosal dendritic cells and their importance in induction of T cell responses we investigated the effect of different S. suis serotype strains and an isogenic capsule mutant of serotype 2 on the maturation, activation and expression of IL-10, IL-12p70 and TNF-α in human monocyte-derived dendritic cells. Additionally, we compared phagocytosis levels and bacterial survival after internalization. The capsule of serotype 2, the most common serotype associated with infection in humans and pigs, was highly anti-phagocytic and modulated the IL-10/IL-12 and IL-10/TNF-α cytokine production in favor of a more anti-inflammatory profile compared to other serotypes. This may have consequences for the induction of effective immunity to S. suis serotype 2 in humans. A shielding effect of the capsule on innate Toll-like receptor signaling was also demonstrated. Furthermore, we showed that 24 h after phagocytosis, significant numbers of viable intracellular S. suis were still present intracellularly. This may contribute to the dissemination of S. suis in the body.     

Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes strains.

Streptococcus pyogenes causes severe invasive diseases in humans, including necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome (STSS). We found that mice infected intramuscularly (i.m.) with S. pyogenes strains developed bacteremia and subsequent sudden death after at least 10 days of a convalescent period. Mostly, it occurred more than 21 days after muscle infection. We provisionally designate this phenomenon as "delayed death." Just after muscle infection, all the mice lost weight and activity, but recovered completely within 3 days. They had kept good activity and a fine coat of fur till one or two days before their death. Some of the dead mice were found to have soft-tissue necrosis. There was no correlation between the virulence leading to the delayed death and the severity of diseases from which strains were isolated. It was also found that the production of neither streptococcal pyrogenic exotoxin (SPE) A nor B correlated to the virulence leading to delayed death. The bacteria obtained from the organs of the mice with delayed death expressed capsule. We suggest that the mice with delayed onset of systemic bacterial dissemination and subsequent death after muscle infection with S. pyogenes are the animal models of STSS, because the pathophysiology is extremely similar to that of human STSS.     

Immunoglobulin M efficacy against Cryptococcus neoformans: mechanism, dose dependence, and prozone-like effects in passive protection experiments

The IgM mAbs 12A1 and 13F1 are protective and nonprotective, respectively, against lethal Cryptococcus neoformans infection in mice. To better understand the variables that contribute to IgM efficacy against C. neoformans, we studied the effects of inoculum size, route of infection, and Ab dose for each of these mAbs. mAb 13F1 did not prolong survival under any condition studied. mAb 12A1 prolonged survival after the administration of certain Ab doses after i.p. infection with defined inocula and promoted phagocytosis, agglutination, and the formation of inflammatory cell rings around yeast cells in vivo. Large Ab doses of mAb 12A1 resulted in either no protection or enhanced infection, consistent with a prozone-like effect. Investigation of this phenomenon revealed that the fungal cell was protected against microbicidal nitrogen-derived oxidants when large amounts of Ab were bound to the C. neoformans capsule. mAb 12A1 was opsonic in vitro for peritoneal, but not splenic or alveolar macrophages. In summary, our results indicate that IgM efficacy against C. neoformans is a function of the route of infection, inoculum, and Ab dose and is associated with its ability to promote opsonization, agglutination, and phagocytic ring formation in vivo. The occurrence of the prozone-like phenomenon implies that high Ab titers are not necessarily beneficial in assuring protection against certain pathogens and that caution should be exercised in using high Ab titer as a measure for vaccine efficacy.     

B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus

West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (microMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, an approximately 500-fold difference in serum viral load was detected in micro MT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected micro MT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.     

The Bps polysaccharide of Bordetella pertussis promotes colonization and biofilm formation in the nose by functioning as an adhesin

Many respiratory pathogens establish persistent infection or a carrier state in the human nasopharynx without overt disease symptoms but the presence of these in the lungs usually results in disease. Although the anatomy and microenvironments between nasopharynx and lungs are different, a virulence factor with an organ‐specific function in the colonization of the nasopharynx is unknown. In contrast to the severity of pertussis and mortality in non‐vaccinated young children, Bordetella pertussis results in milder and prolonged cough in vaccinated adolescents and adults. Individuals harbouring bacteria in the nasopharynx serve as reservoirs for intrafamilial and nosocomial transmission. We show that the Bps polysaccharide of B. pertussis is critical for initial colonization of the mouse nose and the trachea but not of the lungs. Our data reveal a biofilm lifestyle for B. pertussis in the nose and the requirement of Bps in this developmental process. Bps functions as an adhesin by promoting adherence of B. pertussis and Escherichia coli to human nasal but not to human lung epithelia. Patient serum specifically recognized Bps suggesting its expression during natural human infections. We describe the first bacterial factor that exhibits a differential role in colonization and adherence between the nasopharynx and the lungs.     

Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.     

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.     

Zika virus dynamics: Effects of inoculum dose,the innate immune response and viral interference

Experimental Zika virus infection in non-human primates results in acute viral load dynamics that can be well-described by mathematical models. The inoculum dose that would be received in a natural infection setting is likely lower than the experimental infections and how this difference affects the viral dynamics and immune response is unclear. Here we study a dataset of experimental infection of non-human primates with a range of doses of Zika virus. We develop new models of infection incorporating both an innate immune response and viral interference with that response. We find that such a model explains the data better than models with no interaction between virus and the immune response. We also find that larger inoculum doses lead to faster dynamics of infection, but approximately the same total amount of viral production.     

Type 3 Secretion System Cluster 3 Is a Critical Virulence Determinant for Lung-Specific Melioidosis

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD₅₀ of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD₅₀ using IMIT. We subsequently examined the LD₅₀ of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen.     

Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae

Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.     

Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.     

Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus

In the current global AIDS pandemic, more than half of new human immunodeficiency virus type 1 (HIV-1) infections are acquired by women through intravaginal HIV exposure. For this study, we explored pathogenesis issues relevant to the development of effective vaccines to prevent infection by this route, using an animal model in which female rhesus macaques were exposed intravaginally to a high dose of simian immunodeficiency virus (SIV). We examined in detail the events that transpire from hours to a few days after intravaginal SIV exposure through week 4 to provide a framework for understanding the propagation, dissemination, and establishment of infection in lymphatic tissues (LTs) during the acute stage of infection. We show that the mucosal barrier greatly limits the infection of cervicovaginal tissues, and thus the initial founder populations of infected cells are small. While there was evidence of rapid dissemination to distal sites, we also show that continuous seeding from an expanding source of production at the portal of entry is likely critical for the later establishment of a productive infection throughout the systemic LTs. The initially small founder populations and dependence on continuous seeding to establish a productive infection in systemic LTs define a small window of maximum vulnerability for the virus in which there is an opportunity for the host, vaccines, or other interventions to prevent or control infection.     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

Inactivation of the CovR/S Virulence Regulator Impairs Infection in an Improved Murine Model of Streptococcus pyogenes Naso-Pharyngeal Infection

Streptococcus pyogenes is a leading cause of pharyngeal infection, with an estimated 616 million cases per year. The human nasopharynx represents the major reservoir for all S. pyogenes infection, including severe invasive disease. To investigate bacterial and host factors that influence S. pyogenes infection, we have devised an improved murine model of nasopharyngeal colonization, with an optimized dosing volume to avoid fulminant infections and a sensitive host strain. In addition we have utilized a refined technique for longitudinal monitoring of bacterial burden that is non-invasive thereby reducing the numbers of animals required. The model was used to demonstrate that the two component regulatory system, CovR/S, is required for optimum infection and transmission from the nasopharynx. There is a fitness cost conferred by covR/S mutation that is specific to the nasopharynx. This may explain why S. pyogenes with altered covR/S have not become prevalent in community infections despite possessing a selective advantage in invasive infection.