Birgitta Henriques Normark

Intracellular Recognition of Lipopolysaccharide by Toll-like Receptor 4 in Intestinal Epithelial Cells

Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor κB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.

Influenza enhances susceptibility to natural acquisition of and disease due to Streptococcus pneumoniae in ferrets.

The role of respiratory viruses in the transmission of Streptococcus pneumoniae is poorly understood. Key questions, such as which serotypes are most fit for transmission and disease and whether influenza virus alters these parameters in a serotype-specific manner, have not been adequately studied. In a novel model of transmission in ferrets, we demonstrated that pneumococcal transmission and disease were enhanced if donors had previously been infected with influenza virus. Bacterial titers in nasal wash, the incidence of mucosal and invasive disease, and the percentage of contacts that were infected all increased. In contact ferrets, viral infection increased their susceptibility to S. pneumoniae acquisition both in terms of the percentage infected and the distance over which they could acquire infection. These influenza-mediated effects on colonization, transmission, and disease were dependent on the pneumococcal strain. Overall, these data argue that the relationship between respiratory viral infections, acquisition of pneumococci, and development of disease in humans needs further study to be better understood.

Molecular and clinical characteristics of invasive group A streptococcal infection in Sweden

BACKGROUND The incidence and severity of invasive group A streptococcal infection demonstrate great variability over time, which at least, in part, seems to be related to group A streptococcal type distribution among the human population. METHODS An enhanced surveillance study of invasive group A streptococcal infection (746 isolates) was performed in Sweden from April 2002 through December 2004. Noninvasive isolates from either the throat or skin (773 isolates) were collected in parallel for comparison. Clinical and epidemiological data were obtained from 88% of patients with invasive disease and were related to isolate characteristics, including T type, emm sequence type, and the presence of 9 superantigen genes, as well as pulsed-field gel electrophoresis pattern comparisons of selected isolates. RESULTS The annual incidence was 3.0 cases per 100,000 population. Among the patients with invasive disease, 11% developed streptococcal toxic shock syndrome, and 9.5% developed necrotizing fasciitis. The overall case-fatality rate was 14.5%, and 39% of the patients with streptococcal toxic shock syndrome died (P<.001). The T3/13/B3264 cluster accounted for 33% of invasive and 25% of noninvasive isolates. Among this most prevalent type cluster, emm types 89 and 81 dominated. Combined results from pulsed-field gel electrophoresis, emm typing, and superantigen gene profiling identified subgroups within specific emm types that are significantly more prone to cause invasive disease than were other isolates of the same type. CONCLUSIONS This study revealed a changing epidemiology of invasive group A streptococcal infection in Sweden, with emergence of new emm types that were previously not described. The results also suggest that some clones may be particularly prone to cause invasive disease.

Differences in Potency of Intravenous Polyspecific Immunoglobulin G against Streptococcal and Staphylococcal Superantigens: Implications for Therapy of Toxic Shock Syndrome

Administration of intravenous polyspecific immunoglobulin G (IVIG) has been proposed as adjunctive therapy for toxic shock syndrome caused by Streptococcus pyogenes or Staphylococcus aureus. We investigated whether superantigen-containing culture supernatants prepared from streptococcal isolates (n=21) and staphylococcal isolates (n=20) from cases of severe sepsis were inhibited to an equal extent by IVIG in proliferation experiments that used human peripheral blood mononuclear cells. All 3 IVIG preparations tested were highly efficient in neutralizing the superantigens, and most supernatants were completely inhibited at concentrations ranging from 0.05 to 2.5 mg IVIG/mL. An important finding was that culture supernatants from S. pyogenes isolates were consistently inhibited to a greater extent than those of S. aureus isolates (P<.01). The findings demonstrate that staphylococcal superantigens are not inhibited as efficiently as streptococcal superantigens by IVIG, and, hence, a higher dose of IVIG may be required for therapy of staphylococcal toxic shock syndrome in order to achieve protective titers and clinical efficacy.

Myeloid differentiation factor 88-dependent signalling controls bacterial growth during colonization and systemic pneumococcal disease in mice

The Toll‐like receptors (TLRs) and the myeloid differentiation factor 88 (MyD88) are key players in the activation of the innate immune defence during microbial infections. Using different murine infection models,  we  show  that  MyD88‐dependent  signalling  is crucial for the activation of the innate immune defence against Streptococcus pneumoniae. Our data demonstrate that both local and systemic inflammatory response to S. pneumoniae depends on the presence of MyD88 to clear bacterial colonization of the upper  respiratory  tract  and  to  prevent  pulmonary  and systemic infection in mice. Finally, we described a strong correlation between enhanced bacterial growth in the bloodstream of MyD88‐deficient mice and the inability to lower the serum iron concentration in response to infection.

Toxin-Gene Profile Heterogeneity among Endemic Invasive European Group A Streptococcal Isolates

We determined the toxin-gene profiles of 239 endemic, invasive group A streptococcal (GAS) isolates that circulated, within a 5-year period, in European university hospitals. Profiling was performed by use of multiplex polymerase chain reaction that screened for 9 streptococcal pyrogenic exotoxins (speA, speB, speC, speF, speG, speH, speJ, ssa, and smeZ). Analysis revealed that invasive GAS isolates do not share a common toxin-gene profile. Although all emm types were characterized by several different toxin-gene profiles, a predominance of 1 or 2 toxin-gene profiles could be observed, reflecting that a few invasive clones have spread successfully throughout the world. Remarkably, statistical pair-wise analysis of individual toxin genes revealed that strains that did not share the predominant profile still showed a nonrandom distribution of key toxin genes characteristic of the specific emm type. This could indicate that M proteins function, directly or indirectly, as barriers for horizontal gene exchange.

Contribution of host, bacterial factors and antibiotic treatment to mortality in adult patients with bacteraemic pneumococcal pneumonia

Rationale Host and bacterial factors as well as different treatment regimens are likely to influence the outcome in patients with bacteraemic pneumococcal pneumonia. Objectives To estimate the relative contribution of host factors as well as bacterial factors and antibiotic treatment to mortality in bacteraemic pneumococcal pneumonia. Methods A cohort study of 1580 adult patients with community-acquired bacteraemic pneumococcal pneumonia was conducted between 2007 and 2009 in Sweden. Data on host factors and initial antibiotic treatment were collected from patient records. Antibiotic resistance and serotype were determined for bacterial isolates. Logistic regression analyses were performed to assess risk factors for 30-day mortality. Results Smoking, alcohol abuse, solid tumour, liver disease and renal disease attributed to 14.9%, 13.1%, 13.1%, 8.0% and 7.4% of the mortality, respectively. Age was the strongest predictor, and mortality increased exponentially from 1.3% in patients <45 years of age to 26.1% in patients aged ≥85 years. There was considerable confounding by host factors on the association between serotype and mortality. Increasing age, liver disease and serotype were associated with mortality in patients admitted to the ICU. Combined treatment with β-lactam antibiotics and macrolide/quinolone was associated with reduced mortality in patients in the ICU, although confounding could not be ruled out. Conclusions Host factors appear to be more important than the specific serotype as determinants of mortality in patients with bacteraemic pneumococcal pneumonia. Several host factors were identified that contribute to mortality, which is important for prognosis and to guide targeted prevention strategies.

Capsular Expression in Streptococcus pneumoniae Negatively Affects Spontaneous and Antibiotic-Induced Lysis and Contributes to Antibiotic Tolerance

Penicillin and vancomycin induce a lytic response in Streptococcus pneumoniae that requires the N-acetylmuramyl-l-alanine amidase LytA. We show that clinical isolates of pneumococci of capsular serotypes 1, 4, 6B, and 23F were generally less lytic to penicillin than pneumococci of serotypes 14 and 3. In addition, most 9V isolates were less lytic to vancomycin, compared with isolates of other serotypes. Parent-mutant pairs expressing and not expressing capsular serotypes 2, 4, and 9V were compared for antibiotic-induced lysis. The nonencapsulated variants were considerably more lytic after beta-lactam and/or vancomycin treatment, and antibiotic tolerance was seen only in the context of capsule expression. Conversion from a nonlytic to a lytic phenotype, after loss of capsule expression, required an intact lytA autolysin gene. Exogenous addition of purified LytA gave a lower lytic response in capsulated strains, compared with that in nonencapsulated mutants. Spontaneous autolysis in stationary phase also was negatively affected by capsule expression in an autolysin-dependent manner. Long-term starvation in the stationary phase of the vancomycin- and penicillin-tolerant isolate I95 yielded nonencapsulated mutants that had lost antibiotic tolerance and were lytic to penicillin and vancomycin. The 9V capsular locus of I95 and one of these stationary phase-selected mutants were completely sequenced. The only difference found was a 1-bp frameshift deletion in the cps9vE gene of the lytic mutant, encoding a uridine diphosphate-glucosyl-1-phosphate transferase. Two additional independently isolated lytic mutants of I95 from the stationary phase also contained mutations in the same region of cps9vE, which identified it as a mutational hot spot. This report demonstrates that capsular polysaccharides negatively influence the lytic process and contribute to antibiotic tolerance in clinical isolates of pneumococci.

Group A Streptococcal Infections in Sweden: A Comparative Study of Invasive and Noninvasive Infections and Analysis of Dominant T28 emm28 Isolates

Surveillance of group A streptococcus (GAS) infections in Sweden during 1996-1997 indicated that T28 isolates were dominant, whereas T1M1 infections were uncommon. Circulating T28 isolates were nearly all emm28, MLST52, and these clones had also been prevalent 10 years earlier. Isolates from invasive and noninvasive infections were of similar types and prevalences. The average national incidence of invasive episodes was 2.9/100,000 population but varied between 0 and 8.3/100,000 population in different counties. It increased markedly with age, reaching 22.9 episodes/100,000 among people aged > or =90 years. The incidence of puerperal sepsis was higher than expected (22.4/100,000 of those at risk), with 1 death. Overall mortality was 16% and was associated with preexisting chronic disease (P=.002). Streptococcal toxic shock syndrome (STSS) developed in approximately 15% of patients with invasive episodes, with a mortality rate of 45%. The use of nonsteroidal anti-inflammatory drugs was not found to be associated with the development of STSS.

Persistent Infection with Helicobacter Pylori and the Development of Gastric Cancer

Gastric malignancies have been closely linked to infection of the gastric mucosa with Helicobacter pylori, but the individual factors involved in the multistage process of tumor development are still poorly understood. H. pylori evades the host defense system and causes persistent infection and chronic inflammation. Immune activation leads to DNA damage by the release of oxygen and nitrogen radicals. Ongoing tissue repair mechanisms and the secretion of cytokines and growth factors, as well as bacterial effector molecules, cause disturbances in the balance between epithelial cell proliferation and apoptosis, promote the accumulation of potential oncogenic mutations, and support neovascularization and tumor growth. In addition, H. pylori might hamper the development of an efficient antitumor immunity and provoke immune-mediated pathology. This review summarizes the recent progress in the understanding of the intimate bacteria-host relationship and the mechanisms by which H. pylori may promote the process of tumor development.