Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Anna Tomás,Leticia Lery,Verónica Regueiro,Camino Pérez-Gutiérrez,Verónica Martínez,David Moranta,Enrique Llobet,Mar González-Nicolau,Jose L Insua,Juan M Tomas,Philippe J Sansonetti,Régis Tournebize,José A Bengoechea

doi:10.1074/jbc.m114.621292

Abstract

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Highlights

There is limited knowledge of Klebsiella pneumoniae determinants implicated in the blocking of the NF-␬B signaling pathway
None of the metabolic and transport mutants displayed any growth defects under the screening conditions or in LB, making it unlikely that any gross growth defect underlies the activation of NF-␬B in infected cells. As this will be the subject of future studies, at present we speculated that their contribution to Klebsiella immune evasion is indirect and related to their impact on other Klebsiella factors
We discovered that Klebsiella pullulanase T2SS is important for immune evasion in vitro and in vivo

Summary

Conclusion

CPS, LPS, and the T2SS-secreted PulA are needed for immune evasion. Significance: A new therapeutic approach to treat Klebsiella infections will be the prevention of immune evasion. A wealth of evidence indicates that the activation of early inflammatory responses is essential to clear Klebsiella infections [11,12,13,14] Any interference with this response leads to a more severe infection [15], in turn, augmenting the immune response with exogenous inflammatory mediators decreases the mortality associated with K. pneumoniae infection (16 –19). CYLD and MKP-1 are normally regulated to return to homeostasis after inflammation to protect the host from an overwhelming inflammatory response [25, 26] To exert this anti-inflammatory effect, K. pneumoniae affects the membrane association of the receptor NOD1 [22]. Further characterization confirmed the critical role of K. pneumoniae CPS in blocking NF-␬B activation and uncovered the role of the LPS polysaccharide section and the pullulanase type II secretion system (T2SS) in immune evasion

Experimental Procedures

Results

Discussion