Patent Application: US-80774604-A

Abstract:
the invention relates to a mutation within the sap operon of an avirulent clone of a nontypeable strain of haemophilus influenzae . the invention also relates to the nthi sap operon genes and the polypeptides encoded by these polynucleotide sequences . the invention also relates to a novel 110 kda nthi outer membrane protein and the polynucleotide that encodes this outer membrane protein . methods of screening for nthi infection , and treating and preventing nthi related disorders are also contemplated .

Description:
the following examples illustrate the invention wherein example 1 describes construction of a signature - tagged mutagenesis library and identification of avirulent nthi clones , example 2 describes the characterization of the avirulent nthi clone a1 , example 3 describes the in vitro phenotypic characterization of the nthi sapf :: mtn5 mutant , and example 4 describes the omp profile of the nthi sapf :: mtn5 mutant . an attenuated nthi mutant was identified by signature - tagged mutagenesis ( stm ) using the transbullar chinchilla model of om . the nthi , strain 86 - 028np , was mutagenized by minitn5 transposons marked with unique signature tags to construct an stm library . a panel of signature - tagged minitn5 transposons was constructed by cloning an ecori cassette containing the tn903 kanamycin resistance gene into the ecori site and a signature tag sequence into the kpni site within the transposon of the epicentre ez :: tn pmod & lt ; mcs & gt ; transposon construction vector . to ensure that the signature tag sequences give a strong hybridization signal and do not cross hybridize to other tags , the signature tag sequences were screened by dot blot hybridization . to adapt the epicentre minitn5 in vitro transposition mutagenesis system to strain 86 - 028np , single stranded gaps generated by the transposase in the chromosomal dna were repaired using dna polymerase and ligase . the transposon inserted dna was transformed back into the parent strain using m - iv transformation method described in herriott et al . ( j . bacterial . 101 : 513 - 6 , 1970 ). the individual kanamycin resistant clones with unique tags were assembled into 96 well plates for animal screening . southern blot analysis was performed to confirm random and single insertion of the transposon in the stm mutants . a pool of 38 stm mutants containing unique signature tags were directly inoculated into the middle ear cavity of a chinchilla at a concentration of 1 . 0 × 10 6 cfu / ear . the chinchilla was monitored for om development and formation of effusions in the middle ear over a period of 48 hours by otoscopy and tympanometry . effusions were removed by epitympanic taps and plated on chocolate agar plates supplanted with kanamycin to recover the nthi mutants that survived in the middle ear . bacteria recovered after two days of inoculation were selected as the recovery pool , at which time point the proliferation of nthi cells in the middle ear reached a peak level during the course of om development . bacterial genomic dna isolated from the input and recovery pool was used as template for pcr amplification of signature tags . the input and recovery probes were hybridized to membranes spotted with each signature tag pcr product or oligonucleotide in quadruplicate . by comparing the input and recovery hybridization patterns as depicted in fig1 attenuated mutants containing signature tags were identified within the input pool but not in the recovery pool . the mutant carrying the a1 tag ( circled in fig1 ) was cleared from the middle ear in two other independent stm animal experiments confirming that this mutant was attenuated in vivo . this mutant was subjected for further characterization as described below . sequence analysis was carried out on the transposon interrupted dna locus in the attenuated strain using standard methods in the art . southern blot analysis showed that a 6 kb ecorv restricted genomic dna fragment of the mutant of interest contained the transposon interrupted gene . the ecorv restricted genomic dna fragments were cloned into the pbluescript plasmid , and the transposon containing clone , designated pbluea1 , was isolated using marker rescue from lb agar plates supplemented with kanamycin . the 6 kb insert of the pbluea1plasmid was sequenced and the resulting dna sequence data were searched against ncbi databases using the blastx and blastn algorithms . contigs were assembled using seqmanii software ( dnastar inc .). as shown in fig2 sequence analysis indicated that the transposon was inserted 165 bp from the 3 ′- end of the sapf gene , thus this attenuated mutant was designated as “ sapf :: mtn5 .” the coding sequence of the kanamycin resistance gene is in the same orientation as the sapf gene . a search of the h . influenzae rd genome using the sapf dna , identified the haemophilus sap gene cluster containing 6 open reading frames ( orfs ) in the order of sapabcdfz , where the sapf was the fifth gene of the cluster followed by a hypothetical protein h11643 which we designated “ sapz ” in this study . this study , utilized the genomic sequencing nthi strain 86 - 028np and a three - fold coverage contig assembly . part of the sap operon was present in the contigs ( contigs 512 and 324 ; seq id no : 16 - 17 ). a pair of primers were designed according to the contig sequences to pcr amplify the whole sap operon from strain 86 - 028np . sequence comparison analysis showed that the sap operon of strain 86 - 028np had 98 % identity as that of strain rd , and the sap genes were organized in the same way . the polynucleotide sequence of the sap operon genes ( sapa , sapb , sap c , sapd , sapf , and sapz ) are set out as seq id nos : 1 - 6 , respectively . the amino acid sequences of the sap operon gene products , sapa , sapb , sapc , sapd , sapf and sapz , are set out as seq id nos : 7 - 12 respectively . the sapf gene contains an atp - binding domain and may share translocation atpase activity with the sapd gene , shown to be up - regulated in response to iron and may play a role in potassium uptake via the trk system ( harms et al ., microbiology 147 : 2991 - 3003 , 2001 ; paustian et al . j . bacteriol , 184 : 6714 - 20 , 2002 ) the sapz gene is unique to haemophilus . sapz is predicted to be a transmembrane protein with gene homologs in sap operon - containing bacteria , p . multocida , s . typhimurium , s . enterica , and e . coli 0157 : h7 , and in neisseria meningitidis and pseudomonas aeruginosa , which do not contain a sap operon . in bacteria containing the described sap system , however , sapz is not located near the sap operon in the bacterial genome . the nthi sap operon locus is organized as a single operon containing 6 genes as displayed in fig4 and this gene locus was upregulated in vivo as determined by quantitative rt - pcr . dna sequence analysis indicated that the coding sequences of the 86 - 028np 6 sap genes were located on the same dna strand with very few non - coding bases between the orfs ( fig3 ). when the sap gene cluster was scanned for transcriptional terminators ( gcg wisconsin package v . 10 ), one typical rho - independent terminator as a stem - loop structure followed by polyu sequence was found downstream of the sapz gene . therefore , the 6 nthi sap genes were predicted to be organized in an operon structure and presumed to be co - transcribed as one polycistronic mrna . the sapz gene begins 11 nucleotides downstream of the end of the sapf gene and therefore it is highly likely that is co - transcribed with the sap gene cluster . to confirm this organization , rt - pcr was used to determine whether the region between the sap genes was transcribed . each rt - pcr reaction utilized a primer from the 3 ′ end of one gene and a primer from the 5 ′ end of the following gene . if there was a pcr product , the two adjacent genes were cotranscribed . as amplicons were obtained from each junction region , all 6 sap genes were co - transcribed as one polycistronic mrna ( fig3 upper panel ), which was in agreement with the transcriptional property of the sap operon in s . typhimurium ( parra - lopez et al , supra ). in order to determine whether insertion of the transposon prevented transcription of the downstream sapz gene in the sapf :: mtn5 mutant , a similar rt - pcr strategy using primers which annealed to the 3 ′- end of the sapf gene or the minitn5 transposon and a primer which annealed to the 5 ′- end of the sapz gene was employed . as depicted in fig2 both primer sets gave positive results using sapf :: mtn5 rna as template demonstrating that there was detectable sapz mrna produced in the sapf :: mtn5 mutant . the sapz transcript in the mutant is presumably due to the absence of a transcriptional terminator downstream of the kanamycin resistance gene in the minitn5 transposon . thus , the attenuated phenotype of strain sapf :: mtn5 was likely due to the sapf mutation but not the result of polar effect on the downstream sapz gene . to ensure no secondary mutation in the original sapf :: mtn5 mutant contributed to the various phenotypes of this mutant , the parent strain 86 - 028np was transformed with the 6 kb ecorv fragment containing the sapf :: mtn5 allele from the pbluea1 plasmid . the wild type sapf gene was replaced in this strain by homologous recombination with the sapf :: mtn5 allele . one km resistant clone was confirmed to harbor a minitn5 interrupted sapf gene by pcr and southern blot analysis . this clone was further characterized together with the sapf :: mtn5 strain and designated rcsapf :: mtn5 . since the sap mutants of s . typhimurium and e . chrysanthemi were reported to be hypersensitive to certain antimicrobial peptides , sensitivity to several commercial available cationic peptides against the nthi parent and the sapf mutant strains was analyzed . protamine displayed differential killing effect on the sapf mutants comparing to the parent strain . broth minimal inhibitory concentration ( mic ) analyses for protamine determined that the mic of protamine for the sapf :: mtn5 mutants was lower than that for the parent strain ( 0 . 2 mg / ml versus 0 . 4 mg / ml ). growth curve measurement under the same growth condition ( aerobic growth in sbhi broth ) demonstrated that the growth curves of the two mutant strains and the parent strain were identical . this analysis suggests that the two mutant strains do not possess a growth defect . thus , the sapf gene product is not required for growth in enriched media , and the lack of growth of the sapf mutants at the lower protamine concentrations in sbhi broth was not due to a growth defect . therefore , the sapf mutation may be responsible for the phenotype of increased sensitivity to protamine , and the in vivo attenuation property of the sapf mutant . the sapf mutant displayed a minor variation of omp profile in comparison with the parent strain . sarkosyl insoluble omps of the three strains were prepared using differential detergent extraction as described in filip et al ., ( j . bacteriol . 115 : 717 - 722 , 1973 ), and separated in a 10 % sds - page . absence of a 110 kda omp band was consistently observed from several omp preparations in both mutant strains compared to the parent strain . both the original and reconstructed mutant exhibited this minor change of the omp profile , suggesting that the loss of the high molecular protein in the outer membrane was not due to a secondary mutation in the original sapf :: mtn5 mutant . to determine the amino acid sequence of the 110 kda omp protein , a tryptic digest was performed . the 110 kda protein was digested overnight at 37 ° c . subsequently the peptides ( seq id nos : 22 - 39 ) were extracted , desalted ( 10 %) using c18ziptip ( millipore ), and analyzed by matrix - assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof ms ). the peptide information is set out below in table 1 . the maldi monoisotopic peaks were then searched in the ncbinr database using the profound computer program . this analysis identified the 110 kda omp protein as h . influenzae hemoglobin binding protein ( hgba_haein ; genebank accession no . q9kiv2 or closely related homologue ) by the emory microchemical facility . the amino acid sequence from hgba_haein ( q9kiv2 ) ( seq id no : 15 ) was employed to query the 86 - 028np genomic contig set using the tblastn algorithm . the translation of the compliment of nucleotides 2623 to 5358 of contig 516 ( seq id no : 18 ) was a translated sequence that is closely related to amino acids 94 to 1013 of hgba_haein ( seq id no : 15 ). similarly , contig 411 ( seq id no : 19 ) contains nucleic acid sequences whose translation is highly related to amino acids 59 to 148 of hgba_haein and less closely related to amino acids 147 - 969 of hgba_haein . contig 2 ( seq id no : 39 ) contains nucleic acid sequences whose translation is highly related to amino acids 1 to 122 of hgba_haein ( seq id no : 15 ). contigs 469 and 497 ( seq id nos : 20 and 21 ) also contain sequences with homology to hgba_haein . the sequence similarity is summarized in table 2 below . additional sequence analysis identified the full length sequence of the nthi 110 kda omp set out as seq id no : 41 that is encoded by the nucleic acid set out in seq id no : 40 . the sapf gene is 810 base pairs in length ( seq id no : 5 ) and encodes a 269 amino acid protein ( seq id no : 11 ) with a predicted mass of a 30 kda soluble cytoplasmic protein with a an isoelectric point of 6 . 5 . therefore it is unlikely that the biosynthesis or secretion of this 110 kda high molecular mass omp is associated with the sapf gene product . many omps of gram negative pathogens are important virulent factors playing roles in different pathogenesis aspects , such as host cells interaction , adhesion , iron acquisition , antigenic drift . the absence of the 110 kda omp may also contribute to the lost virulence of the sapf : : tn5 mutant . a set of clones with putative promoter activity in vivo were identified by differential fluorescence induction , and upregulated in vivo expression was confirmed by quantitative rt - pcr analysis as described in mason et al . ( infection and immunity 71 : 3454 - 3462 , 2003 ). a clone that contained sequence upstream of the sapa gene was isolated . this clone demonstrated up - regulated gfp fluorescence in vivo indicating increased transcription of the sap operon . sapa was predicted to localize to the periplasm due to its signal sequence and its sequence identity to periplasmic solute binding proteins involved in peptide transport . ( parra - lopez et al ., embo j . 12 : 4053 - 62 , 1993 ) it was predicted that a mutation in the sapa gene would disrupt the function of the sap operon , thereby demonstrating the involvement of sapa in survival in a chinchilla model of otitis media . a non - polar mutation in the sapa gene was generated by insertion of a promoterless kanamycin resistance cassette as described in menard et al . ( j . bacterial ., 175 : 5899 - 906 , 1993 ). the mutant construction was verified by southern blot analysis and the resulting mutant is denoted herein as “ sapa :: kan mutant ”. defensins are known as important elements of innate immunity against microbial infections . in particular , beta - defensins function to protect the host against microbial infections such as gram - negative bacteria infections . recombinant chinchilla beta - defensin - 1 ( cbd - 1 ), an antimicrobial peptide with homology to human beta - defensin - 3 , was used to assess the sensitivity of the sapa :: kan mutant to antimicrobial protection . for microbicidal assays , nthi strain 86 - 028np or its isogenic sapa :: kan mutant were cultured to mid - log phase in brain heart infusion ( bhi ) broth supplemented with 2 μg nad / ml and 2 μg hemin / ml ( sbhi ) or on chocolate agar . static cultures of nthi , s . pneumoniae and e . coli were incubated in 5 % co 2 at 37 ° c . various concentrations of recombinant cbd - 1 ( 2 . 5 , 5 . 0 , 10 . 0 and 20 μg / ml ) were incubated for 1 hour at 37 ° c . in 5 % co 2 with 1 × 10 4 microorganisms in 100 μl of 10 mm sodium phosphate buffer containing either 1 % sbhi . bacteria were serially diluted and plated onto chocolate agar and the cfu of surviving microorganisms per ml was determined following overnight incubation at 37 ° c . in 5 % co 2 . percent killing of the bacteria from a minimum of three replicate assays per strain are presented as mean percent survival (± sd ) relative to concentration of ( r ) cbd - 1 in fig5 . as shown in fig5 the sapa :: kan mutant strain had enhanced sensitivity to killing induced by recombinant chinchilla beta - defensin - 1 as compared to the parental nthi strain . survival of the sapa :: kan mutant was also assessed in vivo . to conduct these studies , a small inoculum of either the parental nthi strain alone , the sapa :: kan mutant alone or a mixture of these two was inoculated into either the nasopharynx or the middle ears of a chinchilla ( chinchilla lanigera ). at periodic time points following inoculation , a nasal lavage or middle ear tapping procedure is done in order to determine the number of bacteria ( in colony forming units per milliliter fluid ) present in each of these anatomic sites within the uppermost airway that are extremely relevant to the disease course of otitis media . in the competitive study wherein the parental nthi strain and the sapa :: kan mutant were mixed in equal parts and inoculated into chinchilla middle ears , as shown in fig6 the ability of the sapa :: kan mutant to survive in the middle ear was dramatically attenuated as compared to the parental strain . the parental strain behaved typically and was present at a very high bacterial load in the middle ears out to eight days after the challenge . in addition , the sapa :. kan mutant was unable to survive when inoculated in the chinchilla middle ear alone as compared to the parental strain inoculated alone . as demonstrated in fig7 in both animals challenged with the sapa :: kan mutant , the bacteria were cleared from both ears of both animals by day 19 or 27 respectively . the parental isolate continued to be culturable at high numbers from the middle ear at these time points ( fig7 ; top panel ). similarly , the sapa :: kan mutant was unable to survive when inoculated alone into the nasopharynx of a chinchilla ( fig7 ; bottom panel ). whereas the parental isolate maintained stable colonization of the nasopharynx , the sapa :: kan mutant was cleared 12 days after challenge . while the present invention has been described in terms of specific embodiments , it is understood that variations and modifications will occur to those skilled in the art . accordingly , only such limitations as appear in the claims should be placed on the invention . glu asn gly leu thr tyr cys thr his ala ser gly phe ser phe asn pro gln thr ala asp ala gly thr ser met asn val val thr glu gln ile tyr asn lys leu phe 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