Differential Candida albicans lipase gene expression during alimentary tract colonization and infection

The human pathogenic fungus Candida albicans, which can reside as a benign commensal of the gut, possesses a large family of lipase encoding genes whose extracellular activity may be important for colonization and subsequent infection. The expression of the C. albicans lipase gene family (LIP1-10) was investigated using a mouse model of mucosal candidiasis during alimentary tract colonization (cecum contents) and orogastric infection. LIPs4-8 were expressed in nearly every sample prepared from the cecum contents and infected mucosal tissues (stomach, hard palate, esophagus and tongue) suggesting a maintenance function for these gene products. In contrast, LIPs1, 3, and 9, which were detected consistently in infected gastric tissues, were essentially undetectable in infected oral tissues. In addition, LIP2 was expressed consistently in cecum contents but was undetectable in infected oral tissues suggesting LIP2 may be important for alimentary tract colonization, but not oral infection. The host responded to a C. albicans infection by significantly increasing expression of the chemokines MIP-2 and KC at the site of infection. Therefore, differential LIP gene expression was observed during colonization, infection and at different infected mucosal sites.     

Pathogenic mechanisms of invasive group A Streptococcus infections by influenza virus–group A Streptococcus superinfection

Group A Streptococcus (GAS) are pathogenic bacteria of the genus Streptococcus and cause severe invasive infections that comprise a wide range of diverse diseases, including acute respiratory distress syndrome, renal failure, toxic shock‐like syndrome, sepsis, cellulitis and necrotizing fasciitis. The essential virulence, infected host and external environmental factors required for invasive GAS infections have not yet been determined. Superinfection with influenza virus and GAS induced invasive GAS infections was demonstrated by our team in a mouse model, after which clinical cases of invasive GAS infections secondary to influenza virus infection were reported by other investigators in Japan, USA, Canada, UK China, and other countries. However, the pathogenic mechanisms underlying influenza virus‐GAS superinfection are not yet fully understood. The present review describes the current knowledge about invasive GAS infections by superinfection. Topics addressed include the bacteriological, virological and immunological mechanisms impacting invasion upon superinfection on top of underlying influenza virus infection by GAS and other bacteria (i.e., Streptococcus pneumoniae and Staphylococcus aureus). Future prospects are also discussed.

     

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.     

Mast cell cathelicidin antimicrobial peptide prevents invasive group A Streptococcus infection of the skin

Mast cells (MC) express cathelicidin antimicrobial peptides that act as broad-spectrum antibiotics and influence the immune defense of multiple epithelial surfaces. We hypothesized that MC help protect against skin infection through the expression of cathelicidin. The susceptibility of MC-deficient mice (Kit Wsh(-/-)) to invasive group A streptococcus (GAS) was compared with control mice. Following s.c. injection of GAS, MC-deficient mice had 30% larger skin lesions, 80% more lesional bacteria, and 30% more spleens positive for bacteria. In contrast to results obtained when GAS was injected into skin, no significant differences were noted between MC-deficient mice and control mice after GAS was applied topically, indicating that MC activity is most important after barrier penetration. To determine whether these differences were due to MC expression of cathelicidin, MC-deficient mice were reconstituted with MC derived from either wild-type or cathelicidin-deficient (Camp(-/-)) mice and challenged with GAS. Forty-eight hours after bacterial injection, mice that did not receive MC had an average lesion size of 200 mm(2), mice reconstituted with wild-type MC showed lesions comparable to control mice (25 mm(2)), while mice reconstituted with Camp(-/-) MC showed an average lesion size of 120 mm(2). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis of cathelicidin peptide purified from mast cells defined this as a unique 28-aa peptide. Combined, these results show that MC confer defense against Gram-positive bacterial infection in the skin, a function mediated in part by the expression of a unique cathelicidin peptide.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection

Group A streptococcal (GAS) pharyngitis and the subsequent bacterial colonization of the human throat elicit an immune response that may precipitate acute rheumatic fever in a susceptible host. To study the bacterial determinants that influence throat colonization and induction of humoral immunity, we characterized the behavior of GAS strains in a baboon model. An M-type 3 clinical isolate of GAS typical of strains that cause pharyngitis and invasive infection was recovered from the pharynx of six out of six baboons for at least 6 weeks after oral inoculation. By contrast, an isogenic mutant deficient in M protein failed to colonize most animals or was rapidly cleared. An isogenic mutant deficient in hyaluronic acid capsule colonized five out of six animals, but only persisted in the pharynx for 14–21 days. Colonized animals developed serum anti- streptolysin O (SLO) and anti-M protein immunoglobulin (Ig)G. The kinetics of the antibody responses were similar to those seen after human infection. Peak titres increased with the duration of throat carriage. Colonization with GAS prevented recurrent colonization after challenge with the homologous wild-type strain, but not after challenge with a strain of different M protein type. Early clearance of the M protein-deficient strain was associated with increased susceptibility of this strain to phagocytic killing in non-immune serum, whereas clearance of the acapsular strain was associated with increased susceptibility to phagocytic killing in the presence of specific antibody. These studies support critical and distinct effects of the GAS M protein and capsule on throat colonization and induction of humoral immunity in a model that reproduces important features of pharyngeal colonization and immune response following human infection.     

Fungal gene expression during pathogenesis-related development and host plant colonization

To successfully infect plants, pathogenic fungi must recognize and communicate with their host during different stages of the disease cycle. In past years, techniques such as insertional mutagenesis, sensitive GFP-based reporter systems and microarray techniques have been developed to analyze these processes at the molecular level, and now novel insights into this fascinating aspect of pathogen-plant communication are beginning to emerge. This is exemplified by a number of pathogenicity genes functioning in distinct stages of pathogenic development in Magnaporthe grisea.     

A locus of group A Streptococcus involved in invasive disease and DNA transfer

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengths-transposon-mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of approximately 10-8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.