From the Section of Infection, Departments of Medicine, Taipei Medical University - Wan Fang Hospital, Taipei, Taiwan;
1Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan.
Received: Mar. 6, 2007; Accepted: Aug. 2, 2007
Correspondence to: Dr. Li-Min Huang, Department of Pediatrics, National Taiwan University Hospital. 7, Jhongshan S. Rd., Jhongjheng District, Taipei City 100, Taiwan (R.O.C.) Tel.: 886-2-23123456 ext. 5139; Fax: 886-2-23934749; E-mail: lmhuang@ha.mc.ntu.edu.tw
The Transforming Streptococcus Pneumoniae
in the 21st Century
Yu-Chia Hsieh, MD, PhD; Wen-Sen Lee, MD; Pei-Lan Shao
1, MD;
Luan-Yin Chang
1, MD, PhD; Li-Min Huang
1, MD, PhD
Streptococcus pneumoniae, an important pathogen causing sepsis, sinusitis, otitis media, bacterial meningitis and bacterial pneumonia, results in global morbidity and mortality each year. The burden of pneumococcal disease is highest in chil-dren and the elderly. Treatment of pneumococcal infection has been hampered by the complexity of the host immune response. In recent decades, the increase of S. pneumoniae strains’ resistance to `-lactam antibiotics and other classes of antimicrobials has made treatment even more complicated. Fortunately, the advent of heptavalent conjugate vaccine con-fers a high degree of protection against pneumococcal disease and colonization caused by vaccine serotype strains. After the introduction of conjugate pneumococcal vaccine, invasive pneumococcal disease caused by vaccine serotypes and antibi-otic-resistant isolates has been reduced. However, naturally
transformable pneumococci may escape vaccine-induced immunity by switching their cap-sular genes to non-vaccine serotypes. Development of cheaper, serotype-independent vac-cines based on a combination of protein antigens should be pursued. (Chang Gung Med J 2008;31:117-24)
Key words: Streptococcus pneumoniae, conjugate vaccine, transformable
S
treptococcus pneumoniae, a pathogen discoveredmore than one hundred years ago, remains a lead-ing cause of bacteremia, sinusitis, otitis media, bac-terial meningitis and pneumonia. This bacterium is present worldwide, and is associated with substantial illnesses and deaths in humans.(1) Historically, study
of the biology of S. pneumoniae led to the identifica-tion of the nature of genetic material, the phenome-non of quorum sensing, the use of polysaccharide-based vaccine and the recognition of bacterial
resis-tance to antimicrobial drugs.(2,3) Since the complete
genome of S. pneumoniae was decoded in 1997, much has been discovered about the bacterial pro-teins involved in pneumococcal disease, the regula-tion of virulence and the regularegula-tion of DNA uptake.(4)
Recently, the landscape of pneumococcal infection has been changed by two major events, namely, availability of conjugate pneumococcal vaccine and more aggressive behavior of pneumococcal pneumo-nia.(5,6)It is now a good time to review our
standing of the biology and clinical behavior of S.
pneumoniae.
S. pneumoniae virulence factors
Capsule
Polysaccharide capsule is the earliest known S.
pneumoniae virulence factor, and serves as a
para-digm for studies of immune responses and polysac-charide biochemistry. Capsular polysacpolysac-charide is composed of multiple sugars that help pneumococci fight against phagocytosis. The amount of capsule expression in the microbe changes during replication in the host, a phenomenon known as phase varia-tion.(7)Reduced capsule expression (transparent
vari-ant) in the nasopharynx is instrumental in exposing the adhesins necessary for colonization, whereas increase in capsule expression (opaque variant) is essential for avoiding complement-mediated opsonophagocytosis during invasive disease. Several factors such as BOX elements, capsule locus A (CpsA), CpsB, CpsC and CpsD, and spontaneous sequence duplication contribute to the complex regu-lation of capsule synthesis.(8-12)
Choline-binding proteins
S. pneumoniae possesses several
choline-bind-ing proteins on its surface that serve as a way of attaching it to the cell surface. The most well-known choline-binding proteins in pneumococci are autolysin, pneumococcal surface protein C (PspC) and pneumococcal surface protein A (PspA). Autolysin (LytA amidase) degrades peptidoglycan of the pneumococcal cell wall and separates daughter cells. Lysis of pneumococci by autolysin leads to release of the pneumococcal cell wall and pneu-molysin, which in turn induce inflammatory respons-es and cause tissue damage.(13) PspA is a protective
antigen of S. pneumoniae, and is able to inhibit com-plement deposition and activation.(14,15)It contributes
to pneumococcal virulence in both bacteremia and sepsis models.(16) PspC, also referred to as
choline-binding protein A (CbpA), acts as an adhesin, and interacts with the polymeric immunoglobulin recep-tor (pIgR) on mucosal epithelial cells to facilitate adhesion and invasion.(17)
Pneumolysin and other virulence factors
The role of pneumolysin in pneumococcal
infec-tion has been well studied. Pneumolysin belongs to the family of pore-forming toxins, which can lyse cell membranes containing cholesterol. This toxin also activates the complement system, induces the production of proinflammatory mediators, recruits inflammatory cells and causes cell apoptosis.(18,19)
Other proteins, including LPXTG-anchored protein (hyaluronidase, neuraminidase and serine protease), lipoprotein, hydrogen peroxide, superoxide dismu-tase, NADH (nicotinamide adenine dinucleotide, reduced form) oxidase, as well as zinc metallopro-tease (immunoglobulin A prometallopro-tease, ZmpB and ZmpC), also contribute to the virulence of S.
pneu-moniae. A pneumococcal pilus encoded by the rlrA
pathogenicity islet, consisting of LPXTG-containing surface proteins and sortases, enhances adherence and stimulates the host inflammatory response.(20)
Pneumococ-cal neuraminidases cleave sialic acid-containing substrates. Neuraminidase A and B both have essential roles in respiratory tract infection and sepsis. Neuraminidase C may contribute to the abili-ty of pneumococci to cause meningitis.(21)
Capsular type or clonal type determine the invasive capacity of S. pneumoniae
S. pneumoniae can be divided into more than 91
distinct types according to capsular polysaccharides but only 20 to 30 types are associated with human diseases. Hence, there is an association between serotype and the potential of pneumococci to cause invasive disease. Certain serotypes, such as serotype 1 are highly invasive, mostly due to the specific chemical composition of their capsules. Serotype 3 can evade the immune system, readily resulting in a fatal disease.(22) Further studies of the population
biology of S. pneumoniae found that, even within the same serotype, some individual clones (such as ST9 and ST124) were significantly overrepresented in invasive diseases compared with carriage.(23) So far,
the exact mechanisms of why some serotypes can go beyond colonization to cause invasive disease remain unclear but it appears that the capsule is not suffi-cient to determine invasive potential or inflammatory response.(24,25)The genetic background of the host, in
addition to the capsule, also plays a critical role in dictating virulence. Understanding the underlying mechanism of virulent genotypes becomes a priority in the era of the pneumococcal conjugate vaccine.
Innate immunity
S. pneumoniae infection is countered by a robust
inflammatory reaction in the host. Complement, C-reactive proteins (CRP), surfactant protein, Toll-like receptors (TLR) and T cells comprise the major com-ponents of the immune response against S.
pneumo-niae. Studies using mice deficient in specific genes
indicated that both the classical and alternative com-plement pathways were vital in host defense against pneumococcal infection.(26)CRP specifically binds to
phosphocholine residues of C-polysaccharide (PnC) in the cell wall of S. pneumoniae to activate the clas-sical pathway of complement in human serum.(27)
Lung surfactant protein-D (SP-D) facilitated the early clearance of pneumococci in a murine model of bronchopneumonia and bacteremia.(28) TLR2
recog-nizes pneumococcal lipoteichoic acid (LTA) and cell wall peptidoglycan to initiate an inflammatory response. TLR2 also had a protective role in sys-temic infection and nasopharyngeal colonization in a murine model.(29,30) TLR4 recognizes pneumococcal
pneumolysin to limit pneumococcal proliferation in the nasopharynx.(31) TLR4 also interacts with
pneu-molysin to induce mammalian cell apoptosis against pneumococcal infection.(32) CD4 (cluster of
differen-tiation 4) T cells were found to contribute to early protective immunity to S. pneumoniae based on stud-ies using mice lacking the major histocompatibility complex II (MHCII) gene.(33)However, how CD4+ T
cells function in this aspect remains unclear.
In addition, Nod1 and Nod2 are cytosolic pro-teins of the pathogen recognition receptor within host cells that respond to pneumococci.(34) The
myeloid differentiation factor (MyD88) is an adaptor molecule in the signaling of the host inflammatory cascade against pneumococcal infection.(35)
Pneumococcal colonization
The first step leading to pneumococcal disease is nasopharyngeal colonization. S. pneumoniae spreads through respiratory droplets. Following exposure, the pathogen may establish itself in the nasopharynx of the new host. The human nasophar-ynx is the only known natural reservoir for S.
pneu-moniae. Invasive pneumococcal disease occurs when
pneumococci gain access into deep human tissues, which might be facilitated by prior virus infection, especially influenza virus infection.(36)S. pneumoniae
invades human nasopharyngeal epithelial cells
through a process termed reverse endocytosis medi-ated by pIgR. Nasopharyngeal colonization is dynamic, and influenced by overcrowding, smoking, ethnicity and socioeconomic status.(37) Colonization
rates vary from 3% to 70% in healthy children in dif-ferent countries and gradually decline with age up to adulthood.(38-40)One way to reduce invasive
pneumo-coccal disease is prevention of colonization. However, this may lead to replacement by other bac-terial species in the nasopharynx, such as
Staphylococcus aureus and Haemophilus influen-zae.(41)Hence, a protein-based pneumococcal vaccine
to prevent the invasive disease without disturbing the bacterial ecology in the nasopharynx may be consid-ered for controlling pneumococcal disease.(41)
Evolution of S. pneumoniae
S. pneumoniae was the first pathogen to
demon-strate the phenomenon of transformation. In 1944, Avery et al. proved that the genetic material in bacte-rial cells was DNA by using a transformation model in S. pneumoniae.(42)Natural competence for genetic
transformation in S. pneumoniae is mediated by a quorum sensing-regulated system. CSP, a heptade-capeptide pheromone, induces competence in grow-ing cells at a critical cell density by activatgrow-ing the 2-component signal transduction system comDE.(3)Due
to the ability to undergo horizontal gene transfer, S.
pneumoniae easily adapts to environmental changes,
which leads to substantial genetic heterogeneity as well as genomic plasticity (Fig. 1). The first example is the presence of divergent mosaic blocks in peni-cillin binding protein (PBP) genes in penipeni-cillin-resis- penicillin-resis-tant S. pneumoniae under the selective pressure of
Fig. 1 Evolution of naturally transformable Streptococcus
pneumoniae.
penicillin pressure evolutionary vaccine pressure pressure PBP in Streptococcus mitis or Streptococcus oralis novel variant to be hyper-virulent capsular switching mosaic PBP in S. pneumoniae
penicillin. Mosaic PBP genes evolve to be penicillin-resistant via acquiring PBP from other Streptococcus species.(43) The second example is the evolution to
greater virulence via recombination. Serotype 6B causes more invasive diseases than serotype 6A. By using multilocus sequence typing, serotype 6B clones evolved almost exclusively by recombination, whereas serotype 6A evolved by mutation.(44) The
third example is capsular switching under a large-scale vaccination program.(45) Although the current
introduction of conjugate pneumococcal vaccine has successfully reduced invasive pneumococcal disease caused by the vaccine serotypes and effectively decreased the spread of antimicrobial drug-resistant isolates, pneumococcal infection remains a major issue. At least two consequences have been noted since the use of heptavalent conjugate vaccine. First, serotypes not covered by the conjugate vaccine have increased both in nasopharyngeal colonization and clinical illness.(45) Second, serotype switching can
occur through recombination in naturally trans-formable clones and result in the acquisition of a non-vaccine capsule to escape vaccine-induced immunity.(45) Furthermore, the ability of different
serotypes to be transformed affected the evolutionary biology and genetic diversity of each serotype. Serotype competence accounts for why the reported serotypes that underwent in vivo capsular transfor-mation were also antibiotic-resistant. Gene transfer has been a powerful tool in the evolution of S.
pneu-moniae.
Emerging disease: complicated pneumonia
S. pneumoniae is the most common pathogen of
pyogenic pneumonia in children. Previous studies have shown that the lungs return to normal after pneumococcal pneumonia, regardless of the severity at the peak stage of the disease. This is for two rea-sons. First, S. pneumoniae usually induces granulo-cyte apoptosis, which tends to limit tissue injury and promotes the complete resolution of pneumonia.(46)
Second, S. pneumoniae produces few exotoxins capable of inducing lung damage, in contrast to other organisms such as Staphylococcus aureus and
Streptococcus pyogenes, which produce a variety of
tissue-damaging substances causing lung necrosis and destructive lung injury.(47)Since the advent of the
use of penicillin, S. pneumoniae infection has rarely developed into empyema or lung necrosis.
However, an increase of complicated pneumo-coccal pneumonia, including necrotizing pneumonia, lung abscess and empyema, has been observed in children since the 1990s(5,48,49) (Table 1). The
occur-rence of complicated pneumonia was associated with longer durations of fever, longer oxygen requirement and longer hospital stays.(5,48) Older age, white race,
presence of immature polymorphonuclear leukocytes in peripheral blood, high CRP levels, no underlying disease or chest pain on presentation were predictors of lung necrosis and/or abscess.(5,48) This increase of
Table 1. Studies of an Increase in Complicated Pneumococcal Pneumonia
Reference Country Year Pattern of complicated pneumonia Prevalent serotype (5) U.S.A. 1993-2000 necrotizing pneumonia, pleural effusion, empyema and lung abscess 14, 1, 19, 6, 3
(50) U.S.A. 1996-2000 empyema 1, 14, 6B, 19F
(pre-PCV7)
2001-2005 empyema 1, 3, 19A
(post-PCV)
(51) U.K. 1997-2001 empyema 1, 14, 3
(54) U.K. 1997-2003 cavitary disease 1, 3, 14, 9V
(48) Taiwan 1995-2003 necrotizing pneumonia, empyema 14, 23, 19, 9 (49) Israel 1986-1997 pleural effusion, empyema, pneumothorax, pneumatocele and/or Not done
atelectasis
(55) Singapore 1997-1999 cavitary necrosis, abscess formation, empyema Not done
(56) Spain 1993-2003 parapneumonic pleural effusion Not done
complicated pneumonia is not directly related to the increase in penicillin-resistant S. pneumoniae.(5,48,49)In
the U.S., serotype 14 was the most common serotype causing complicated pneumonia, whereas serotype 1 and serotype 3 significantly caused complicated pneumonia compared to those serotypes causing lobar pneumonia in children before the widespread use of heptavalent pneumococcal conjugate vac-cine.(5) After the utilization of conjugate vaccine,
serotype 1 remained prevalent, and serotypes 3 and 19A were increasingly detected.(50) In the U.K.,
serotype 1 was also the dominant serotype causing pneumococcal empyema.(51) Clonal spread of
pneu-mococcal serotype 1 is speculated to contribute to the increased complicated pneumonia in the U.S. and U.K. Interestingly, serotype 1 S. pneumoniae was rare in the nasopharynx but had a high clinical inci-dence. This serotype was common in both Northern Europe and North America in the early 20th century, and now has become more prevalent in developing countries such as Rwanda, Egypt and Africa. Poverty, overcrowding and decreased availability of antibiotics all contribute to the spread of serotype 1.(52) In view of the rare carriage of serotype 1 S.
pneumoniae, it is mysterious as to how it is
transmit-ted among humans. In most cases of culture-negative parapneumonic pleural effusion or empyema, serotype 1 was the frequent etiology.(51,53)
Surprisingly, several studies failed to identify serotype 1 in clinical samples in Taiwan. Instead, the major clone associated with complicated pneumonia in Taiwan was serotype 14.(48)Since serotype 1 is
dif-ficult to culture, whether there is real serotype differ-ence in complicated pneumonia is worth further study in Taiwan.
Conclusion
Given the proclivity of horizontal gene transfer, current advances in antimicrobial therapy and serotype-limited conjugate vaccine are inadequate to combat pneumococcal diseases. In the future, better understanding of molecular interaction at the cellular level could provide insight into the development of protein vaccine and new modulation therapy.
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