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Infection and Immunity, December 2009, p. 5572-5582, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00831-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Regulated Delayed Expression of rfaH in an Attenuated Salmonella enterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen{triangledown}

Qingke Kong, Qing Liu, Kenneth L. Roland, and Roy Curtiss III*

Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, Tempe, Arizona 85287

Received 24 July 2009/ Returned for modification 20 August 2009/ Accepted 23 September 2009

RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A {Delta}rfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, {Delta}PrfaH178, was introduced into the attenuated vaccine strain {chi}9241 ({Delta}pabA {Delta}pabB {Delta}asdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic {Delta}rfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent Streptococcus pneumoniae WU2. Immunization with {Delta}PrfaH178 mutant strains led to increased levels of protection compared to that of the parent {chi}9241 and of a {Delta}rfaH derivative of {chi}9241.

* Corresponding author. Mailing address: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, P.O. Box 875401, 1001 S. McAllister Avenue, Tempe, AZ 85287-5401. Phone: (480) 727-0445. Fax: (480) 727-0466. E-mail: rcurtiss{at}asu.edu

{triangledown} Published ahead of print on 5 October 2009.

Editor: B. A. McCormick

Infection and Immunity, December 2009, p. 5572-5582, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00831-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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