These studies demonstrate unequivocally that the T2SS of P. aeruginosa
is capable of causing death in lung infections, an issue that has been unsettled. They also demonstrate that the onset of action differs between the T2SS and the T3SS , with the T3SS acting rapidly within 24 h, and the T2SS causing death at a slower rate, differences that were not hitherto appreciated and no doubt contributed to the lack of an appreciation of the role of the T2SS. It is not known whether this difference in the timing of lung injury is due to the fact that activation of the T2SS requires that bacteria reach high concentrations to achieve quorum sensing [26
] or due to the fact that pathologic lesions are late in appearing; however, because the bacterial count in the lungs had increased significantly by 24 h, the observations are consistent with a role of quorum sensing in mediating lung injury and death.
These studies also narrow down the possible pathways used for T2 toxin secretion. It has long been suspected that the Xcp secreton was responsible for secretion of the better-known T2 secreted toxins of P. aeruginosa
], but the discovery of the Hxc secreton [7
] has raised the possibility that this pathway may also be used for novel toxin secretion. Additionally, there is XqhA [8
], an alternative secretin that enables low-level secretion of XcpQ substrates. Neither of these, however, appear to play significant roles in virulence in this model, because deletion of xcpQ
alone renders the organism avirulent in the absence of T3 secretion.
The ability of P. aeruginosa
to avoid phagocytic clearance by PMNs is a major virulence determinant. Successful evasion of phagocytosis by P. aeruginosa
is believed to be primarily dependent on the presence of a functional T3SS, because this system has been demonstrated to kill neutrophils [28
]. However, it also appears that a functional T2SS causes a clearance defect, as demonstrated in the present study by rising bacterial counts when the T2SS is present and more viable bacteria within the neutrophils. The sole possible insight into this defect involved the elevated concentrations of the anti-inflammatory cytokine interleukin-10 in BAL fluid. This cytokine has been demonstrated to impair both neutrophil and alveolar macrophage bactericidal activity [29
], and antibody against it improves survival in P. aeruginosa
lung infection in a cecal ligation puncture model of immunosuppression [25
]. Thus, there may be 2 independent actions of the T2SS as a whole that are not fully explained by our knowledge of the secreted products—a toxic effect on the mouse lung and an independent one on host defenses. In vitro, none of the major T2 secreted enzymes (ExoA, LasB, and phospholipase H) kill neutrophils [31
], even though subtle effects on function have been reported [32
]. In vivo, a mutant in a regulatory gene for ExoA, has been shown to impair host defenses in the lungs of wild-type mice by allowing bacterial counts to reach higher levels, but this mutation did not affect survival of mice and it had no effect on neutrophil migration into the airways [33
]. Elucidating which toxins are having these effects(s) will require a detailed characterization of the secreted products that use the XcpQ secretin and testing of mutants in the secreted products.
It may be argued that the T3SS is the first system to act, and because it kills more rapidly, there may not be a role for the T2SS. However, not all P. aeruginosa
strains are competent for T3 secretion. Sokol et al [34
] examined 124 clinical isolates from burns and bacteremia and found that only 38% of the isolates secreted ExoS, whereas 80% secreted ExoA. Assuming that strains having ExoU were missed (approximately one-third of those that are ExoS positive), this suggests that only half of these strains were T3 secretion competent. Roy-Burman et al [10
] examined 71 non CF lung isolates and found that only 66% secreted ExoS or ExoU. Hauser et al [35
] examined 35 selected strains from patients with pneumonia and found that 74% were T3SS competent, but they further pointed out that among these same isolates, secretion of ExoS was not consistently associated with virulence in a mouse model of pneumonia [36
], suggesting that other virulence factors played a role in death. We have also examined 100 blood isolates for T2 secretion, using elastolytic activity as a proxy for T2 secretion, and have found that 99% of these isolates are T2 secretion competent (data not shown). Thus, these 2 systems may be viewed together as comprising a fail-safe system for defense against whatever host the organism encounters and are both integral to pathogenesis, neither more important than the other.
The findings in this study are also of practical significance in the development of active or passive vaccines or treatments for P. aeruginosa
if one wishes to target the toxin-producing systems. Targeting either the T3SS or the T2SS, or any of their products alone, will not be optimal. Although it has been demonstrated that targeting PcrV of strain PA103 is protective [37
], it should be pointed out that this strain demonstrates defects in T2 secretion; it does not secrete the major protease LasB and lacks a flagellum [38
] and is therefore not representative of a large number of P. aeruginosa
strains. Thus, some surface-exposed component of the T2SS or a specific toxic secretion product needs to be included in a vaccine designed to target secretions.
Besides demonstrating a role for the T2SS, this study also demonstrates the potential of using TLR-knockout mice to elucidate pathogenesis under circumstances where there is host compromise. A reasonable assumption is that for a significant infection caused by this opportunistic bacterium [39
] to occur there must be failure of innate immunity which we have replicated by using these mice. Although challenge with larger numbers of bacteria will overcome the innate immune response of normal mice, this is also likely to cause an inflammatory response that may confound an analysis of the effects on individual toxins, especially if multiple toxins act together to cause death.