Facultatively attenuated bacterial species and methods of preparation and use thereof

Abstract

The present invention provides facultatively attenuated bacterial species and methods of preparation and use thereof. The term “facultatively attenuated” as used herein refers to a bacterium which comprises a set of defined recombinant modifications which have substantially no effect on the ability of the bacterium to grow by multiplication when the bacterium is outside of its host organism, but which result in deletion of one or more genes essential for multiplication of the bacterium when the bacterium is introduced into its host organism, for example within host cells of a vaccinate recipient. These recombinant modifications take advantage of regulatory sequences which preferentially induce expression of genes within the mammalian host.

We claim: 
     
         1 . A bacterium comprising:
 (i) a nucleic acid encoding a recombinase heterologous to the bacterium operably connected to regulatory sequences which preferentially induce expression of the recombinase when the bacterium is in a mammalian host, and   (ii) a first attachment site for the recombinase introduced upstream from one or more genes in the bacterial genome which are essential for multiplication of the bacterium, and a second attachment site for the recombinase introduced downstream from the gene(s) genes in the bacterial genome which are essential for multiplication of the bacterium, wherein the first and second attachment sites are configured to provide recombinase-catalyzed removal of the one or more genes in the bacterial genome which are essential for multiplication of the bacterium upon expression of the recombinase.   
     
     
         2 . A bacterium according to  claim 1 , wherein the bacterium is a facultative intracellular bacterium, and the regulatory sequences preferentially induce expression of the recombinase when the bacterium is in a mammalian host cell. 
     
     
         3 . A bacterium according to  claim 1 , wherein the bacterium is of a genus selected from the group consisting of  Listeria, Neisseria, Mycobacterium, Francisella, Bacillus, Salmonella, Shigella, Yersinia, Brucella, Legionella, Rickettsia , and  Chlamydia.    
     
     
         4 . A bacterium according to  claim 1 , wherein the bacterium is  Listeria monocytogenes.    
     
     
         5 . A method according to  claim 4 , wherein the  Listeria monocytogenes  is ΔActA/ΔInlB. 
     
     
         6 . A bacterium according to  claim 4 , wherein the regulatory sequences comprise a  Listeria monocytogenes  promoter which is PrfA-dependent. 
     
     
         7 . A bacterium according to  claim 6 , wherein PrfA-dependent promoter is selected from the group consisting of the inlA promoter, the inlB promoter, the inlC promoter, the hpt promoter, the hly promoter, the plcA promoter, the mpl promoter, and the actA promoter. 
     
     
         8 . A bacterium according to  claim 7 , wherein the PrfA-dependent promoter is an actA promoter. 
     
     
         9 . A bacterium according to  claim 1 , wherein the recombinase is selected from the group consisting of φC31 integrase, R4 integrase, TP901 integrase, φBT1 integrase, B×B1 integrase, PSA integrase, Cre recombinase, Flp recombinase, XerC recombinase, λ integrase, HK01 integrase, P1 integrase, HP1 integrase, L5 integrase, γδ recombinase, Tn3 recombinase, gin recombinase, RV integrase, SPBc integrase, TG1 integrase, φC1 integrase, MR11 integrase, φ370 integrase, φK38 integrase, Wβ integrase, and BL3 integrase. 
     
     
         10 . A bacterium according to  claim 1 , wherein the first attachment site is an attB site and the second attachment site is an attP site. 
     
     
         11 . A bacterium according to  claim 1 , wherein the one or more genes essential for multiplication of the bacterium comprise at least one gene involved in DNA replication. 
     
     
         12 . A bacterium according to  claim 11 , wherein the at least one gene involved in DNA replication is selected from the group consisting of ori, dnaA, dnaN, gyrA, gyrB, polC, dnaE, ftsK, ftsZ, ligA, dnaG, parC, parE, holB, dnaX, SMC, and ftsY. 
     
     
         13 . A bacterium according to  claim 1 , wherein the one or more genes essential for multiplication of the bacterium are part of an operon, and the first attachment site is upstream of all or a portion of the operon, and the second attachment site is downstream of all or a portion of the operon. 
     
     
         14 . A bacterium according to  claim 13 , wherein the first attachment site is upstream of the operon, and the second attachment site is downstream of the operon. 
     
     
         15 . A bacterium according to  claim 1 , wherein the recombinase is a Cre recombinase, the regulatory sequences comprise an actA promoter, the first and second attachment sites comprise loxP sites, and the gene(s) essential for multiplication of the bacterium flanked by the first and second attachment sites comprise the bacterium's origin of replication. 
     
     
         16 . A bacterium according to  claim 15 , wherein the bacterium is  Listeria monocytogenes.    
     
     
         17 . A bacterium according to  claim 1 , wherein the first and second attachment sites flank a nucleic acid sequence about 20 kb in length or less. 
     
     
         18 . A bacterium according to  claim 17 , wherein the nucleic acid sequence is 10 kb in length or less. 
     
     
         19 . A bacterium according to  claim 18 , wherein the nucleic acid sequence is about 6 kb in length. 
     
     
         20 . A bacterium according to  claim 1 , wherein the bacterium comprises within the bacterial genome an exogenous nucleic acid sequence encoding a heterologous polypeptide, wherein the exogenous nucleic acid sequence is operably connected to regulatory sequences which preferentially induce expression of the heterologous polypeptide when the bacterium is in a mammalian host.