Compositions and methods for non-antibiotic treating of bacterial infections by blocking or disrupting bacterial genes involved in virulence or viability

Abstract

Provided are modified Staphylococcus aureus pathogenicity islands of a variety of types and origins. The pathogenicity island are characterized as having a polynucleotide comprising a bacterial pathogenicity island nucleotide sequence (a “B-PINS”) that contains one or more modifications (“modified B-PINS”), that include i) a deletion or disruption of at least one virulence determinant of the B-PINS; and ii) an insertion of at least one cargo sequence. The polynucleotide are capable of being packaged within a bacterium into a phage-like particle that comprises a bacteriophage capsid, tail and tail fiber proteins in the form of an antibacterial drone (“ABD”). The ABD is capable of infecting bacteria such that at least one cargo sequence is introduced into bacteria infected by the ABD. Pharmaceutical and other compositions the ABDs are also included. Bacteria that make the ABDs are provided, as are isolated and/or purified ABDs. Methods of killing or otherwise modifying bacteria using the ABDs are also provided.

What is claimed is: 
     
         1 . A polynucleotide comprising a bacterial pathogenicity island nucleotide sequence (B-PINS) comprising one or more modifications (a modified B-PINS),
 wherein the one or more modifications comprise:   i) a deletion or disruption of at least one virulence determinant of the B-PINS; and   ii) an insertion of at least one cargo sequence into the B-PINS;   wherein the polynucleotide comprising the modified B-PINS is capable of being packaged within a bacterium into a phage-like particle that comprises or consists of bacteriophage capsid, tail and tail fiber proteins, and wherein the phage-like particle (an antibacterial drone “ABD”) is capable of infecting bacteria such that the at least one cargo sequence is introduced into bacteria infected by the ABD.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the one or more modifications of i) comprises a deletion of all or substantially all of the virulence determinants in the B-PINS. 
     
     
         3 . The polynucleotide of  claim 1 , wherein the at least one cargo sequence comprises a polynucleotide sequence encoding an anti-bacterial agent that is functional against a target in at least some bacteria in a bacterial population. 
     
     
         4 . The polynucleotide of  claim 3 , wherein the anti-bacterial agent comprises one or a combination of:
 a) a sequence encoding an RNA or DNA polynucleotide that comprises specificity for the target, wherein the target comprises an essential gene or an RNA encoded by an essential gene or a variable gene or an RNA encoded by a variable gene;   b) a sequence encoding a protein that has specificity for the target and can participate in and/or cause its cleavage and/or inactivation; or   c) a sequence encoding a toxin that is cytostatic against bacteria, or a non-toxin protein that inhibits growth of bacteria in the bacterial population or inhibits formation of and/or kills persister bacteria and/or dormant viable but non-culturable (VBNC) bacteria.   
     
     
         5 . The polynucleotide of  claim 4 , wherein a) comprises an anti-sense RNA. 
     
     
         6 . The polynucleotide of  claim 4 , wherein b) comprises a Clustered Regularly Interspaced Short Palindromic repeats (CRISPR) guide RNA targeted to a sequence in the bacteria in the bacterial population. 
     
     
         7 . The polynucleotide of  claim 4 , wherein the encoded protein of b) comprises a CRISPR-associated nuclease including but not limited to a Cas9 nuclease or a Cpf1 nuclease, or a dCas9. 
     
     
         8 . The polynucleotide of  claim 4 , wherein the toxin of c) comprises a toxin that kills bacteria that have been infected by the ABD. 
     
     
         9 . The polynucleotide of  claim 8 , wherein the toxin comprises lysostaphin. 
     
     
         10 . The polynucleotide of  claim 4 , wherein the toxin of c) comprises a toxin that is secreted by the bacteria and which is bactericidal against the bacteria and bacteria that are uninfected by the ADB. 
     
     
         11 . The polynucleotide of  claim 4 , wherein the target is comprised by a bacterial chromosome or a bacterial plasmid. 
     
     
         12 . The polynucleotide of  claim 10 , wherein the target confers antibiotic resistance. 
     
     
         13 . An ABD of  claim 1 , wherein the polynucleotide in the ABD further comprises a deletion of SaPI genes cpmA and/or B. 
     
     
         14 . A pharmaceutical composition comprising an ABD of  claim 13 . 
     
     
         15 . A method comprising administering a pharmaceutical or other composition comprising ABDs of  claim 13  to an individual in need thereof, wherein subsequent to the administration bacteria in the individual are killed or exhibit reduced virulence. 
     
     
         16 . The method of  claim 15 , wherein prior to the administering, the method further comprises determining a characteristic of bacteria in the individual, and wherein the ABD is designed to comprise a cargo that has specificity for the characteristic. 
     
     
         17 . The method of  claim 16 , wherein the characteristic comprises a nucleotide sequence in the bacteria. 
     
     
         18 . The method of  claim 17 , wherein the nucleotide sequence comprises a sequence that can be recognized by a CRISPR system. 
     
     
         19 . The method of  claim 15 , wherein the ABD comprises a sequence encoding a toxin that is cytostatic against bacteria, wherein the toxin is optionally lysostaphin. 
     
     
         20 . A method for producing an ABD of  claim 13  comprising growing bacterial cells comprising a polynucleotide comprising a bacterial pathogenicity island nucleotide sequence (B-PINS) comprising one or more modifications (a modified B-PINS),
 wherein the one or more modifications comprise: 
 i) a deletion or disruption of at least one virulence determinant of the B-PINS; and 
 ii) an insertion of at least one cargo sequence into the B-PINS; 
 wherein the polynucleotide comprising the modified B-PINS is packaged within the bacteria to thereby produce the ABD. 
 
     
     
         21 . The method of  claim 20 , further comprising separating the ABD from the bacteria. 
     
     
         22 . A bacterial population comprising bacteria which comprise a polynucleotide of  claim 1 . 
     
     
         23 . The bacterial population of  claim 22 , wherein the population comprises a single distinct bacterial strain. 
     
     
         24 . Cell culture media comprising the bacterial population of  claim 22 . 
     
     
         25 . A bacterial population comprising ABDs of  claim 13 . 
     
     
         26 . A method comprising infecting a population of bacteria of with one or more ABDs of  claim 13 , thereby producing a gene drive effect whereby the cargo sequence carried by the one or more ABDs is disseminated to a plurality of bacteria in the population. 
     
     
         27 . A method comprising determining a target sequence in bacteria in a sample, generating or selecting an ABD of  claim 13 , and contacting the source of the sample with a plurality of the generated or selected ABD. 
     
     
         28 . A B-PINS of  claim 1 , wherein the B-PINS comprises a modified  Staphylococcus aureus  pathogenicity island (SaPI). 
     
     
         29 . The B-PINS of  claim 28 , wherein the SaPI is SaPI1, SaPI2, SaPIbov1, or SaPIbov2. 
     
     
         30 . A library of distinct ABDs of  claim 1 , wherein ABDs in the library comprise distinct nucleic acid sequences encoding distinct cargo.