Bacterial interference of ubiquitination and deubiquitination.

Ubiquitination and deubiquitination regulate several essential cellular processes such as protein degradation, cell-cycle progression, signaling, and DNA repair. Given the importance of these processes, it is not surprising that many microbes have developed the means to interfere with different stages of ubiquitin pathways to promote their survival and replication. This review focuses on virulence proteins of bacterial pathogens that mediate these effects and summarizes our current understanding of their actions.

SseL, a Salmonella deubiquitinase required for macrophage killing and virulence.

Expression of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system is controlled by the two-component regulatory system SsrA-SsrB. We used a transcriptomic approach to help define the SsrA-SsrB regulon. We identified a gene encoding an uncharacterized effector (SseL) whose translocation into host cells depends on the SPI-2 secretion system. SseL has similarities to cysteine proteases with deubiquitinating activity. A GST-SseL fusion protein specifically hydrolyzed mono- and polyubiquitin substrates in vitro with a preference for K63-linked ubiquitin chains. Ubiquitin-modified proteins accumulated in macrophages infected with Salmonella sseL mutant strains but to a lesser extent when infected with bacteria expressing active protein, demonstrating that SseL functions as a deubiquitinase in vivo. Salmonella sseL mutant strains did not show a replication defect or induce altered levels of cytokine production upon infection of macrophages but were defective for a delayed cytotoxic effect and were attenuated for virulence in mice.

Polynucleotide phosphorylase negatively controls spv virulence gene expression in Salmonella enterica.

Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.

SlyA regulates function of Salmonella pathogenicity island 2 (SPI-2) and expression of SPI-2-associated genes.

During the systemic phase of murine infection with Salmonella enterica serovar Typhimurium, bacterial virulence is correlated with the ability to grow and survive within host macrophages. Salmonella pathogenicity island 2 (SPI-2), encoding a type three secretion system, has emerged as an important contributor to Salmonella intracellular growth. SPI-2 mutants have been proposed to be more accessible than wild-type Salmonella to oxyradicals generated by the NADPH phagocyte oxidase. We performed mixed infections of mice to investigate the relationship between SPI-2 and SlyA, a transcriptional regulator that confers resistance to oxyradicals. In mixed-infection experiments, the SPI-2 null mutant was severely attenuated in virulence, whereas slyA mutants were only mildly attenuated. Surprisingly, further experiments indicated that the function of SPI-2 was partially dependent on slyA. The intracellular behavior of a slyA mutant in infected cells was consistent with inefficient SPI-2 expression, as formation of Salmonella-induced filaments and the intracellular F-actin meshwork, features that depend on SPI-2, were present at abnormally low frequencies. Furthermore, the translocated levels of the SPI-2 effector SseJ were severely reduced in a strain carrying a mutation in slyA. We used flow cytometry to investigate the role of SlyA in expression of green fluorescent protein (GFP) from transcriptional fusions with promoters of either of two other SPI-2 effector genes, sifB and sifA. The slyA mutant exhibited reduced GFP expression from both promoters. Combining mutations in slyA and other regulators of SPI-2 indicated that SlyA acts through the SsrAB two-component regulatory system. SlyA exhibits partial functional redundancy with OmpR-EnvZ and contributes to the transcriptional response to low osmolarity and the absence of calcium, two environmental stimuli that promote SPI-2 gene expression.

Human-like immune responses in CD46 transgenic mice.

Neisseria meningitidis is a major cause of sepsis and/or meningitis. These bacteria normally cause disease only in humans, however, mice expressing human CD46 are susceptible to meningococcal disease. To explain the sensitivity of CD46 transgenic mice to meningococci, we evaluated early immune responses. Stimulation of TNF, IL-6, and IL-10 was stronger in CD46 transgenic mice compared with nontransgenic mice, and resembled human responses. In CD46 transgenic mice, bacterial clearance in blood started at later time points, and neutrophil numbers in blood were lower compared with nontransgenic mice. Further, elevated levels of activated microglia cells and cyclooxygenase-2 were observed in brain of infected CD46 transgenic mice. Intraperitoneal administration of meningococci lead to increased levels of macrophages only in the i.p. cavity of CD46 transgenic mice. Most of the responses were impaired or absent using LPS-deficient meningococci, showing the importance of LPS in the early immune response to meningococcal infection. Taken together, these data demonstrate that responses in mice expressing human CD46 mimic human meningococcal disease in many aspects, and demonstrate novel important links between CD46 and the innate immune system.

Neisseria meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

Neisseria meningitidis colonizes the upper respiratory tract (URT), enters the blood stream, and reaches the cerebrospinal fluid (CSF). In the present study, we show that bacteria isolated from the URT adhere better to human epithelial cells, compared with bacteria from blood or CSF, which suggests that important changes of virulence-associated proteins take place during bacterial dissemination. Phase variation in the pilus adhesin PilC and sequence variation in the pilus subunit PilE occurred among strains from 1 patient. Changes were not found in the invasion-associated opacity proteins or in lipooligosaccharides. PilC was frequently expressed in serogroup B strains and in URT strains but was often switched off in other serogroups and in CSF strains. Strains lacking PilC showed impaired adhesion to epithelial cells. These data argue that N. meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

CD46 in meningococcal disease.

The human-specific bacterial pathogen Neisseria meningitidis is a major cause of sepsis and/or meningitis. The pili of N. meningitidis interact with CD46, a human cell-surface protein involved in regulation of complement activation. Transgenic mice expressing human CD46 were susceptible to meningococcal disease, because bacteria crossed the blood-brain barrier in these mice. Development of disease was more efficient with piliated bacteria after intranasal, but not intraperitoneal, challenge of CD46 transgenic mice, suggesting that human CD46 facilitates pilus-dependent interactions at the epithelial mucosa. Hence, the human CD46 transgenic mice model is a potentially useful tool for studying pathogenesis and for vaccine development against meningococcal disease.