BACTERIOPHAGE 2016,London

 

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Bacteriophage Therapy: An Alternative Strategy to Combat Drug Resistance

Mycobacteriophage D29

By coderet (Jemboss software).
coderet extracts the coding nucleotide sequence (CDS), messenger RNA nucleotide sequence (mRNA),non- coding nucleotide sequence  and translations specified by the feature tables of the input sequence(s).
By input of D29 genome file.gbk in coderet ( 49136 bp  DNA  linear download from:
ftp://ftp.ncbi.nih.gov/genomes/Viruses/),
the result is:

   CDS     mRNA    non-c    Trans       Total        Sequence
 =====   =====     =====   =====     =====       ========
    79            0            156          79            235          NC_001900


From this work  ( and my contributions):

"D29 was first isolated from soil, and shown to be active against Mycobacterium tuberculosis (Froman et al., 1954).

The plaques produced on the tubercle bacillus were clear, indicating that D29 is a lytic phage (i.e. incapable of lysogeny).

Studies of D29 suggest that it is a member of the "L5-like'' family of mycobacteriophages.

The overall organization of the D29 genome is similar to that of L5, although a 3.6 kb deletion removing the repressor gene accounts for the inability of D29 to form lysogens.

L5 genome

D29 genome

Although lytic, D29 is subject to superinfection immunity by L5; in other words, it is incapable of infecting an L5 lysogen of M. smegmatis.
Only the product of L5 gene 71 is required to prevent infection of M. smegmatis by D29, demonstrating that this is true immunity rather than exclusion (Donnelly-Wu et al., 1993).

L5 gene 71

-The protein  sequence of L5 gene 71 is extracts by SnapGene software:

 -By JEmboss software  the predicting secondary structure of Immunity repressor protein:

a-there is one  helix-turn-helix nucleic acid binding motif:

b-the Aa composition of the protein by L5 gene 71:

c-no presence of transmembrane segments in  this protein
 
d-no site of cleavage between a signal sequence and this protein

e-4 poor PEST motifs as potential proteolytic cleavage sites:

Poor PEST motif with 24 amino acids between position 159 and 183.

   159 HTNLTAEGELLWSWPDDIEELLSEP 183

       PEST score: 3.07 

Poor PEST motif with 23 amino acids between position 117 and 141.

   117 RDDDLVLEFDPSIEPYEGMAGGGFR 141

       PEST score: -3.93 

Poor PEST motif with 11 amino acids between position 62 and 74.

    62 RQIVQQNWPWDTR 74

       PEST score: -16.67 

Poor PEST motif with 11 amino acids between position 16 and 28.

    16 RIPLTLSEIEDLR 28

       PEST score: -10.33 

 
In addition to the shared immunity of L5 and D29, these phages appear to have a common mode of entry in M. smegmatis.
In particular, overexpression of the M. smegmatis mpr gene confers resistance to both L5 and D29, but not to other mycobacteriophages (Barsom & Hatfull, 1996).

The identity of the receptor used for adsorption of these phages is not clear,although pyruvylated, glycosylated acyltrehaloses have been implicated in the infection of M. smegmatis by D29 (Besra et al., 1994).

The two phages have similar host ranges (our unpublished observations), though there are some differences in infection requirements (Fullner & Hatfull,1997), and D29 has been reported to adsorb to Mycobacterium leprae (David et al., 1984).

We therefore assessed the morphologies of L5 and D29 by electron microscopy and found then to be virtually identical (Figure 1).

We have also compared the virion proteins of D29 and L5 by SDS-PAGE of whole particles (Figure 2). The patterns are similar but not identical.

In particular,we note that D29, like L5, has several protein species larger than 150 kDa (bands A, B, and C). In L5, this reflects the presence of extensive covalent crosslinking between subunits of the major capsid protein, gp17 (Hatfull & Sarkis, 1993). Presumably, the major head subunit of D29 is also extensively crosslinked. Indeed, amino-terminal sequencing of the D29 equivalent of band C revealed that it is the product of gene 17 (data notshown).

Many of the L5 proteins (including bands F, H,and J) are represented in similar sizes in D29,although proteins D and E are either absent or in low abundance in D29 (Figure 2). Additionally, a band corresponding to the L5 band I appears to be missing in D29. Instead, D29 has a protein band that migrates only slightly faster than band H. Finally, the intensity of band F is lower in D29 than in L5. In L5, band J corresponds to the major tail subunit protein gp23 (Hatfull & Sarkis, 1993).
Sequencing of the amino-terminal end of the D29 equivalent of band J (data not shown) revealed that the first ten amino acids of this protein are identical to the first ten amino acids of L5 gp23, and also match the predicted N-terminal sequence of D29 gp23. Therefore, gp23 most likely constitutes the major tail subunit of D29.

The G+C content of the D29 genome is 63.6%, a value consistent with that of the mycobacterial hosts (Clark-Curtiss,1990), and virtually identical to the
G+C content of L5 (63.2%; Hatfull & Sarkis, 1993).

 Sequencing directly from D29 DNA we found that the D29 chromosome contains cohesive termini identical to those found in L5 (5'-GGTCGGTTA-3' with a 3' extension; data not shown).

 Although D29 is not a temperate phage, it does contain a phage attachment site (attP) that is very similar to L5's, including the common core (although one position at the extreme end is different such that only 42 bp are common to the M. smegmatis attB sequence; see PenÄa et al., 1997).

 The D29 attachment site (coordinates of the common core are 26,634 to 26,676) is located somewhat to the right of center of the genome and divides the genome into a left and right arm as described for L5 (Figure 3).

While the D29 and L5 genomes are closely related, the level of similarity is
not the same across the genomes. In particular, the left arms can be aligned without introducing substantial gaps and are approximately 80% identical at the nucleic acid level. The right arms are considerably less closely related although the differences are not evenly distributed. Thus, in the right arm, regions of high similarity are punctuated by segments of unrelated DNA (see below).

We identified three database matches for D29 gene products for which there is no homolog in L5. One of these is an internal segment of gp10 
(which is absent from L5) that matches HI1415, a hypothetical protein of
unknown function from  Haemophilus influenzae Rd (Fleischmann et al., 1995).
The remainder (genes 36.1 and 59.2) have reasonable similarities to deoxycytidinylate deaminases and non-heme haloperoxidases, respectively.

 Although D29 is a close relative of temperate L5 and has an attP-integration system, it does not form lysogens. The simplest explanation for D29's exclusively lytic lifestyle is a 3.6 kb deletion relative to L5 at the right end of its genome which removes part of the repressor gene (71) and several adjacent genes (Figure 4).

Alignment of the genome sequences indicates that D29 has lost 3620 bp relative to L5 (between L5 coordinates 44,700 and 48,321; the junction of the flanking sequences in D29 occurs between coordinates 45,706 and 45,707) probably through a simple deletion without substantial additional rearrangements (Figure 4A).

Deletion derivatives of L5 which have lost all or part of the repressor gene (71) and are incapable of forming lysogens have also been isolated (Donnelly-Wu et al., 1993).
By comparing the L5 and D29 coding regions at the point of deletion,
it is apparent that D29 has lost about half of both genes 71 and 82 and
presumably all of the intervening genes (Figure 4B). In L5, gene 71 encodes 
the phage repressor, and is required for the maintenance of lysogeny and is sufficient to confer superinfection immunity (Donnelly-Wu et al., 1993).

Checking by Gepard software and Mauve software 

 
The observation that D29 does not have an intact copy of gene 71 thus completely explains its lytic properties. In addition, because D29 presumably has lost L5 genes 71 to 82, this region is most likely not essential for phage growth, an observation consistent with previous reports that the entire region from 71 to 88 is non-essential (Sarkis et al., 1995). Little is known about the functions of any of the genes in this area of the L5 genome, although a gene in the 72 to 82 interval is potentially involved in lysogenic establishment (Sarkis et al., 1995).

Considerations about Mycobacteriophage D29 that can be used potentially in Buruly therapy

I consider  Mycobacteriophage D29 as point of reference and by the information below  I will start again in the analysis of these mycobacteriophages: D29, L5,TM4,BXZ2 and DS6A (specific only for Mycobacterium tuberculosis complex).

1-D29  is capable to lyse and  to grow on Mycobacterium ulcerans

2- D29  is a L5likevirus,where the potential receptors are located in the tail fibers

 3-D29 is a good model for the question regarding the type of receptor and the differences among the receptors because it is a lytic phage that infects both fast and slow- growing  mycobaterial species

 4- DS6A  is capable to lyse and to grow only on  mycobacteria belonging to Mycobacterium tuberculosis complex  and this property give me the possibility to analyse the differences with D29.

 DS6A phage (my old idea) 

 From this scientific work:

From this scientific work :
" In 1981 Sula et al. reported positive indicators with a reduction in the observed lesions in the spleen, lungs and livers of guinea pigs following therapy with DS6A. More recent work demonstrated that phage therapy could have a beneficial effect in guinea pigs with disseminated tuberculosis, but that its action was considerably less pronounced than that of isoniazid monotherapy."

From this scientific work:
"Sula et al. [63] infected guinea pigs with M. tuberculosis and then treated them with subcutaneous injections of three different bacteriophages twice weekly for 10 weeks.
One of these bacteriophages, designated DS6A, produced an antibacterial effect at least as good as isoniazid."

From this scientific work:
"In addition, the previously isolated phage DS6A, the only one of the 138 phages that does not infect M. smegmatis was sequenced and annotated." 

 

Mycobacteriophages D29,L5,Bxz2 and TM4

From this work

"This phage proved to be lytic in M. ulcerans species which originated from four different continents. M. ulcerans shows genetic heterogeneity and variable phenotypic characteristics among strains of different geographic origins (Portaels et al., 1996)."

" Additionally, phage therapy could be considered for M. ulcerans infections. In Buruli ulcer, mycobacteria are typically found extracellularly, where they might be immediately accessible by lytic phages."

"Although mycolactone is a major component of the cell surface of M. ulcerans, it does not seem to be involved in the binding of phages or the injection of phage DNA, since the avirulent mycolactone-negative mutant 1615M showed the same host range as the wild-type strain."

" Interestingly, none of the tested phages formed plaques on two strains of M. marinum. In the case of TM4 and D29 at least, this was most likely due to an intracellular inhibition of phage replication, rather than a receptor–receptor binding protein mismatch, since shuttle phasmids based on these two phages are able to transfect M. marinum (Rybniker et al., 2003)."

"Host range in mycobacteria other than M. ulcerans and M. marinum

Except for Bxz2, none of the recently isolated phages formed plaques on M. tuberculosis or M. bovis BCG, showing that phages isolated in the fast-growing M. smegmatis are often confined to this mycobacterium or other fast-growing species. It is likely that environmental samples, for example from compost, may contain additional phages, and the use of slow-growing species such as M. bovis BCG should be considered when searching for wild-type phages with a broader or different host spectrum."

 Questions

All these phages grow on Mycobacterium ulcerans and on Mycobacterium smegmatis but do not grow on Mycobacterium marinum.

-The question is why?

- Have all these phages the same receptor for all mycobacteria host range or they have for each mycobacterium species a different receptor?
-Is there an intracellular inhibition of phage replication on M.marinum?

Two conflicting Viewpoints

From:

Bacteriophage Ecology
Population Growth, Evolution, and Impact of Bacterial Viruses

EDITED BY
STEPHEN T. ABEDON

Chapter 13Interaction of bacteriophages with animals (Carl R. Merril)

“13.3 THE FATE OF PHAGES ADMINISTERED TO ANIMALS
...Rapid elimination of phages from the circulatory system is due to functions associated with the organs of the reticulo-endothelial system, primarily the liver and spleen.”

“13.3.1 Phage interactions with the adaptive immune system

When a specific phage strain is first inoculated into an animal or human, if there are no detectable pre-existing antibodies for that specific phage strain, then it is defined as a neoantigen. Whether a phage strain serves as a neoantigen within a given individual depends on the phage-exposure and physiological history of that individual.
...The presence of adaptive antibodies can have significant effects on the pharmacokinetics of phage experiments and/or therapy.”

“13.3.3 Fate of orally administered phages

It should be noted that some investigators who have administered phages orally report the subsequent detection of phages in the circulatory system (Dabrowska et al., 2005a; Görski et al., 2006). While these findings are compatible with the finding of a trace amount of phage in the spleen, liver, and kidney in the germ-free animal experiments, it may be difficult, as previously noted (Section 13.3.2), to achieve the titers needed for some acute infection therapies by the oral route. This concern with limitations of oral phage administration was reinforced by the observations that when healthy adult volunteers were administered T4 phage in their drinking water ,neither T4 phages nor T4-specific antibodies were found in the serum of the volunteers by the end of the study (Bruttin and Brüssow, 2005)”.

From:

BACTERIOPHAGES Biology and Applications

EDITED BY
Elizabeth Kutter
Alexander Sulakvelidze

Chapter 14 Bacteriophage Therapy in Humans

14.5.2.3. The Development of Phage-Neutralizing Antibodies

“...various parameters affect the development of phage neutralizing antibodies. Levels of phage-neutralizing antibodies are low after a single injection or after a series of closely spaced injections, and most of the antibody that develops under these regimes is of the IgM type, whose neutralizing activity is low and largely reversible (Gachechiladze, Chapter 3 box and personal communication).
High titers of long-lasting, higher-affinity IgG antibodies are seen only when multiple injections are spaced several weeks apart. Thus, the production of neutralizing antibodies (i. e., antibodies against the tail adhesins) should not be a significant obstacle during initial or relatively short-term therapeutic treatments, at least. Furthermore,the antigenic properties vary considerably between different phage families; for example, T4 phage is a good immunogen, but T1 and T5 phages are much less antigenic, and a Bacillus subtilis phage reportedly needs an adjuvant and repeated intraperitoneal injections to generate a detectable immune response (Adams, 1959a).”

14.5.2.1. Phage Movement between Biological Compartments
“Indeed, more recent reports have shown that in the presence of host bacteria, therapeutic phages can be found in the mammalian circulatory system irrespective of the administration route; e.g., therapeutic phages administered orally to infected patients were recoverable from their bloodstream for several days (Babalova et al., 1968; Weber-Dabrowska et al., 1987). Also, studies with experimentally infected animals (Bogovazova et al.,1991; 1992) found that phages enter the bloodstream within 2 to 4 hours, and that they are still recoverable from the internal organs (liver, spleen, kidney, etc.) after ca. 10 hours postinjection.”

Does Phage go in or does Phage not go in through the Blood?

Two conflicting Viewpoints


From The Bacteriophages.org

1-The distribution of phage pfu's in mice following various routes of administration

2-Graphic representation of data from the 1943 infectious disease model in which mice were inoculated by intracerebral injection of the bacteria Shigella dysenteriae (at an LD50 level) were compared with uninfected control mice.Graph

All of the mice in this experiment were injected with 10^9 pfu of phage i.p. which was administered at the same time as the bacterial inoculation. The bacteriophage level in the blood of the uninfected animals was compatible with the dilution of the phage concentration in the total fluid volume of the mouse and the lower levels in the brain reflect the relatively smaller blood content in the brain. However, in the infected animals the phage particles are observed to increase at the site of the infection, the brain, while the blood levels of phage appear to be a 'reflection of the events occurring in the brain.

Graphical representation of data presented by Smith and Huggins. (In this set of experiments all of the animals received an intracerebral inoculation of 5X10^2 cfu of an E. coli K1).

The animals treated with phage were injected with 3X10^8 pfu of phage intramuscularly (into the gastrocnemius muscle) at the same time as the bacterial inoculation. These graphs were derived from the data published in tables 9 and 10 in their paper.

Smith, H. W., and M. B. Huggins. 1982. Successful treatment of experimental E.

coli infections in mice using phage: its general superiority over antibiotics. J. Gen.

Microbiol.128:307-318.

What are the main Features for selecting Bacteriophages for Phage Therapy?

We must consider:

Phage Host range

(a mathematical criterion is required for selecting an useful phage among a lot of phages)

A selected Phage must have a Wide Host Range.

A bacteriophage with a wide host range is a phage capable of killing the highest number of different isolated cultures belonging to a given bacterial species ( example: Staphylococcus aureus from various sources).

For this reason it is indispensable to apply a mathematical criterion for selecting a phage among a lot of phages capable of effectively killing bacteria of a targeted bacterial species from various sources.

Virulence

A bacteriophage is selected as “Virulent “ when this phage is capable of effectively killing bacteria from various sources compared to capacity of the non selected phages.

For selecting a phage, by this property, we use the criterion of the lower concentration capable of killing bacteria compared to concentrations of the non selected phages.

Example:

two Phages with the same Host range

Phage A is effective in killing bacteria at 10^7 dilution
Phage B is effective in killing bacteria at 10^9 dilution

Phage B is the selected phage.

Mutant Phages

Selection of a Mutant Phage by Mutagenization:

a) phages must be always lytic phages

b) mutant phages, if compared to wild-type phage ( non mutaginezed phage or Parental Phage), may have both host range and virulence increased.

How many Types of Phage we must use for a Phage Therapy Treatment?

Phages are "bacterium specific" and it is therefore necessary in many cases to take a swab from the patient and culture it prior to treatment.
Occasionally, isolation of therapeutic phages can typically require a few months to complete, but clinics generally keep supplies of phage cocktails for the most common bacterial strains in a geographical area.The host specificity of phage therapy may make it necessary for clinics to make different cocktails for treatment of the same infection or disease because the bacterial components of such diseases may differ from region to region or even person to person.

In addition, due to the specificity of individual phages, for a high chance of success, a mixture of phages is often applied. This means that 'banks' containing many different phages are needed to be kept and regularly updated with new phages, which makes regulatory testing for safety harder and more expensive.

Phages in practice are applied orally, topically on infected wounds or spread onto surfaces, or used during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage,and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

Phages can usually be freeze dried and turned into pills without materially impacting efficacy. 
In pill form temperature stability up to 55°C, and shelf lives of 14 months have been shown.Oral administration works better when an antacid is included, as this increases the number of phages surviving passage through the stomach.

Topical administration often involves application to gauzes that are laid on the area to be treated.Other forms of administration can include application in liquid form. These vials are usually best kept refrigerated. 
The lytic bacteriophages available for phage therapy are best kept refrigerated but discarded if the pale yellow clear liquid goes cloudy.

Phage therapy is generally considered safe. 
As with antibiotic therapy and other methods of countering bacterial infections, endotoxins are released by the bacteria as they are destroyed within the patient (Herxheimer reaction). This can cause symptoms of fever.

Care has to be taken in manufacture that the phage medium is free of bacterial fragments and endotoxins from the production process.

Monomicrobic Infection caused by one Bacterium

For example: a Mix of 3 lytic Phages for Bacterium A

Phage 1
Phage 2
Phage 3


Phage Receptor Types and Receptors Number (?) on the cell wall, example for 3 Phages
(3 different Receptors):Receptor 1(X?), Receptor 2(Y?), Receptor 3(Z?)


Rate of Mutation to Resistance to Phages:Phage 1(10^-7), Phage 2(10^-7), Phage 3(10^-7)

Rate of Mutation to Resistance to all lytic Phages for Bacterium A:
(10^-7)*(10^-7)*(10^-7)=10^-21

When, for example, a priori we suspect an Infection caused by 3 different Bacteria: Bacterium A, Bacterium B, Bacterium C

For example: a Mix of 6 (or 9) lytic Phages (2 or 3 Phages for each bacterium):

For Bacterium A
Phage A1
Phage A2

For Bacterium B
Phage B1
Phage B2

For Bacterium C
Phage C1
Phage C2


Phage Receptor Types and Receptors Number (?) on the cell wall, example for 6 Phages (2 different Receptors for each bacterium ):

Bacterium A
Receptor A1(X1?)
Receptor A2(X2?)

Bacterium B
Receptor B1(Y1?)
Receptor B2(Y2?)

Bacterium C
Receptor C1(Z1?)
Receptor C2(Z2?)



Rate of Mutation to Resistance to lytic Phages for each bacterium:
 
Bacterium A
Phage A1 (10^-7)
Phage A2 (10^-7)
 
Bacterium B
Phage B1 (10^-7)
Phage B2 (10^-7)
 
Bacterium C
Phage C1 (10^-7)
Phage C2 (10^-7)


Rate of Mutation to Resistance to lytic Phages:

Bacterium A =(10^-7)*(10^-7)= 10^-14
Bacterium B =(10^-7)*(10^-7)= 10^-14
Bacterium C =(10^-7)*(10^-7)= 10^-14