- Volume 152, Issue 1, 2006

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

The phoU gene is the last cistron in the pstSCAB–phoU operon and functions as a negative regulator of the Pho regulon. The authors previously identified a phoU mutant of extraintestinal pathogenic Escherichia coli strain CFT073 and demonstrated that this mutant was attenuated for survival in the murine model of ascending urinary tract infection. It is hypothesized that the PhoU protein might serve as a urovirulence factor by indirectly affecting the expression of virulence-related genes. In this study, the phoU mutant was further characterized and PhoU was confirmed as a virulence factor. Western blot analysis demonstrated that insertion of the transposon in the phoU gene disrupted the expression of PhoU. The phoU mutant had derepressed alkaline phosphatase activity under phosphate-excess and -limiting conditions. In single-challenge murine ascending urinary tract infection experiments, quantitative cultures of urine, bladder and kidney revealed no significant differences between the phoU mutant strain and the wild-type strain CFT073. However, in competitive colonization experiments, the phoU mutant strain was significantly out-competed by the wild-type strain in the kidneys and urine and recovered in lower amount in the bladder. Complementation of the phoU mutant with a plasmid containing the wild-type phoU gene restored the expression of PhoU and alkaline phosphate activity to wild-type levels and no significant difference in colonization was observed between the phoU mutant containing the complementing plasmid and wild-type in competitive colonization experiments. In human urine, the phoU mutant and wild-type grew comparably when inoculated independently, indicating that the attenuation observed was not due to a general growth defect. However, as observed in vivo, the wild-type out-competed the phoU mutant in competition growth experiments in human urine. These data indicate that PhoU contributes to efficient colonization of the murine urinary tract and add PhoU to a short list of confirmed urovirulence factors.

The evolution of host specialization in pathogens is a topic of considerable interest, particularly since it can represent a decisive step in the emergence of infectious diseases. Aspergillus flavus is an opportunistic fungus capable of infecting a wide variety of hosts, including plants, insects and mammals, although with low virulence. Here the derivation of an A. flavus strain that exhibits severe host restriction is reported. This strain exhibited a severe diminution or a complete lack of conidial production on a variety of standard agar media and on various plant species. However, it retained its ability to infect insects from various orders and to re-emerge from and adequately conidiate on the insect cadavers as a culmination of the pathogenic life cycle. This strain, demonstrating insect-dependent conidiation, was discovered to be a cysteine/methionine auxotroph due to an inability to reduce sulfate to sulfite. However, other A. flavus auxotrophs tested for plant and insect host range failed to show insect-dependent conidiation. An association between this specific auxotroph and a decreased host range is shown, emphasizing the role of nutrition in the host–pathogen relationship with respect to host restriction and evolution towards obligate pathogenesis.

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. In Mycobacterium tuberculosis the α-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of the acr gene in M. tuberculosis H37Rv (Δacr strain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesis in vivo was unknown. This study demonstrated that infection of C57BL6 mice with Δacr results in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or Δacr results in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in Δacr-infected mice and was associated with clusters of macrophages within lung granulomas. Δacr-infected mice also showed high serum levels of TNF-α, IFN-γ and G-CSF, suggesting that Acr may play a role in modulating the host response to infection.

Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0·03±0·01 g l−1 for gelatin-based buffered peptone water, compared to 0·21±0·01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 μM tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1·20 g tyrosine l−1, and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.