To our surprise, the sequence of MM1-1998 phage genome was extremely similar to that of MM1. Previously, several isolates of the multidrug-resistant Spain23F-1 clone were shown to contain phage DNA with a highly similar restriction profile (12
). The host of the original MM1, Spain23F-1 strain 949, was isolated in Barcelona, Spain, in 1989. The host of MM1-1998, a penicillin-susceptible type 24 strain, was isolated in March 1998 in Lisbon, Portugal. Between these isolation dates and places lie almost a decade and 1,000 km. Theoretically, a single phage transmission may have happened between the two strains, with the prophage remaining dormant during the rest of the time. Contamination can be excluded, since we never had MM1 in our laboratory and the only Spain23F-1 isolate in our collection does not contain MM1.
Currently, a further MM1 variant can be identified in the unfinished sequence of a strain of the Spain23F-1 clone on the Sanger Institute's website (http://www.sanger.ac.uk/Projects/S_pneumoniae/
). This pneumococcal strain (ATCC 700669) was isolated in 1984 in Spain. The total length of that phage is 39,307 nucleotides, which is 941 bp shorter than MM1. Alignment with MM1 and MM1-1998 showed that this difference is again due to the length of orf47
, which contains two-and-a-half repeats of the 471-bp sequence coding for collagen-like G-X-Y amino acid repeats, i.e., two fewer than MM1. Other than the deletion, the MM1 phage in the Sanger Institute sequence shows 15 single-nucleotide changes compared with MM1. All of these are in the 36 nucleotide changes that distinguish MM1-1998 from MM1. Together, the three sequences show that MM1 prophage changes very little over time in the pneumococcal population despite switching hosts.
There have been few publications dealing with pneumococcal bacteriophages over the last 30 years. Pneumococcal bacteriophages were discovered in the 1970s, when phage studies led to new tools for molecular microbiology and genetics. Some difficulties that were encountered early with pneumococcal phages were unstable stocks, inconsistent plaques, variety of host range, and the apparent nontransformability of lysogens, which was later contested by the addition of external competence-stimulating peptide (20
). The recent finding that lysogenic bacteriophages are an integral part of many bacterial genomes, appear to be responsible for interstrain genetic variation, and carry known or putative virulence factors has revived interest in phages. In pneumococci, however, lysogenic phages are extremely frequent but have not yet been shown to contribute to genetic variation, and no virulence factors have so far been associated with them. This study used in vitro lysogenization and the creation of three isogenic strain pairs to show that the presence of the prophage MM1-1998 induces modifications that may favor colonization or virulence.
The interpretation of the results suggests that MM1-1998 exerts a different influence on the serotype 3 strains than on the other two. Growth speed of the lysogenized and encapsulated type 3 strain 7M was reduced. Adhesion to plastic and both human cell lines was significantly improved in this strain compared to that of the phage-cured 7MC, and in the case of the adherence to cells, this was clearly due to the shutting down of the capsule, since there was no difference when the rough versions 7R and 7RM were compared. The switch to the rough phenotype required the presence of the bacteriophage and contact with the cells and either serum or albumin. Point mutations and sequence duplications in cap3A
(also called cap3D
) have been shown to be responsible for capsule-negative serotype 3 variants (2
). We found a new mutation in cap3A
which was identical in all rough variants of strain 7, suggesting the possibility of a recombination rather than a point mutation. Our results correlate with previous reports of type 3 capsule suppression in the context of adherence: an increase in rough colonies of three serotype 3 strains has been reported in Sorbarod-grown pneumococcal biofilms (38
), and a recent publication showed downregulation of type 3 capsule during contact with cultured lung cells (13
). However, in our study, capsule shutdown during adherence was clearly much more frequent when the MM1-1998 prophage was integrated. Cure rates in both lysogenized mucoid and rough serotype 3 mutants 7M and 7RM were very high (up to 65%). There was, however, an additional difference in transparency of the mucoid colonies (a very watery appearance) coinciding with lysogeny.
In the other two serotypes, 23 and 4, results showed increased adhesion of the lysogenized mutants to plastic and increased adherence of T4M only to pharyngeal cells, as well as no difference for either serotype on bronchial cells. We believe that the absence of a difference on pharyngeal cells between 8 and 8C may be due to the presence of possibly two more prophages in those strains. T4M and T4C are the best characterized strain pair among the three, with T4M containing only one phage, and they seem to show cell type specificity in the adherence assays. The capsule did not appear to be involved in types 23 and 4, since the observed phenotypes on agar, 90% domed/opaque and 10% flat/transparent, were present before and after adhesion, and there was no selection for the flat colony type. Less visible variations of capsule expression were not studied. Spontaneous cure rates of strains 8 and T4M were 1 to 10% in various conditions, much lower than of serotype 3 but still surprisingly high. High phage cure rates may be part of a regulatory system of S. pneumoniae, which supports lysogeny as long as it is useful during one stage of the life cycle but promotes curing when keeping a particular phage becomes unfavorable. We hypothesize that different effects of the phage on type 3 in contrast to types 4 and 23 depends to some extent on the capsule type, or possibly, since they included slower growth speed and very high phage cure rates, a regulatory mechanism that comprises type 3 capsule control.
Our investigation led to the discovery that lysogeny was associated with increased transparency. Transparent colonies, as first described by Weiser et al., appear more collapsed/flat than opaque colonies, and they show earlier autolysis but have identical doubling times and chain length (40
). They were subsequently shown to possess greater amounts of teichoic acids and phosphorylcholine (14
), produced more autolysin and less PspA, appeared to adhere better to platelet-activating factor-transfected COS cells, and colonized rat pups more efficiently (11
). The underlying mechanism responsible for the many changes in phase variation remains unknown. We examined whether lysogeny with MM1-1998 caused transparency or vice versa. We discovered that MM1-1998 produced the same number of plaques in three TIGR4 variants of different transparency. This observation alone does not establish whether one phenotype was infected by more phage particles than the other. It appeared, however, that the choice of a lytic or lysogenic pathway after infection was influenced by the phase of the bacterium and that a selection for transparency may have happened at this stage during the search for lysogens. Plaques on the very opaque Tigr4OL were larger and clearer, suggesting the lytic infectious pathway is favored over the lysogenic in the opaque-phase phenotype. This is supported by the infrequency of lysogens found at the edge of such a plaque. Studies with phage λ have shown that both the physiology of the host cell (growth in poor medium) and multiple-phage infection favor the lysogenic pathway (15
). The very high cure rate of lysogenized Tigr4OL suggests that the opaque phase not only favors the lytic pathway but that it is also incompatible with stable lysogeny after it has been established.
After adjusting for the effect of transparency alone by comparing T4M to an equally transparent Tigr4T in this study, the difference in adherence to plastic and pharyngeal cells remained independently correlated to the presence of the MM1-1998 prophage.
Scanning electron micrographs of strains T4M and T4MC adhering to PVC coupons showed short piliform structures extending from both strains but no structural difference associated with the presence of MM1-1998 prophage. Two tail proteins of the Streptococcus mitis
lysogenic bacteriophage SM1 have been shown to improve adherence of the bacteria to human blood platelets, and they were found to be surface associated. Since none of them had cell wall anchoring or transport signal features, it was hypothesized that they reach the surface either by phage lysis of a subset of cells or by the action of a holin without lysis (4
). A similar mechanism is possible for MM1-1998-mediated adherence. Preliminary comparison of surface protein preparations of T4M and T4MC did not reveal different profiles (data not shown). However, the increased spilling of hemolytic activity into the culture supernatant of lysogenized strains in early stationary phase supports the above hypothesis of phage protein translocation by lysis. Further mutagenesis studies of MM1-1998 genes may shed more light on how the prophage influences adherence. Previously, descriptions of MM1 and similar phages have focused on antibiotic-resistant pneumococci, where they are frequently but not invariably found (8
). Our results showed that antibiotic resistance was not linked to the presence of MM1-1998 prophage.
The present study is the first attempt to lysogenize pneumococcal strains in vitro with a known bacteriophage in order to study the role of the phage in pathogenesis. We discovered that adherence to inert surfaces and specifically to pharyngeal cells is associated with the MM1-1998 prophage, which may confer an advantage in colonization of the human nasopharynx. MM1-like phages are frequent and have been described in multiresistant, globally spread clones (12
). Adherence enhancement would contribute to the fitness of those strains and possibly to their persistence and spread. We furthermore found that the variation between transparent and opaque phases likely plays a role in successful lysogeny of MM1-1998, suggesting that the host bacterium can somewhat control its prophages.