Streptococcus pneumoniae still causes severe morbidity and mortality worldwide, especially in young children and the elderly. Much effort has been dedicated to developing protein-based universal vaccines to conquer the current shortcomings of capsular vaccines and capsular conjugate vaccines, such as serotype replacement, limited coverage and high costs. A recombinant live vector vaccine delivering protective antigens is a promising way to achieve this goal. In this review, we discuss the researches using live recombinant vaccines, mainly live attenuated Salmonella and lactic acid bacteria, to deliver pneumococcal antigens. We also discuss both the limitations and the future of these vaccines.  Streptococcus pneumoniae is the most common cause of pneumonia as well as a number of invasive diseases, such as meningitis and sepsis, and non-invasive mucosal diseases, such as otitis media and sinusitis. It causes severe morbidity and mortality worldwide, especially in young children and the elderly [S. pneumoniae [S. pneumoniae strains worldwide posed another threat to the treatment of infection [S. pneumoniae, pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV) [S. pneumoniae has at least 94 serotypes with different abilities in nasopharyngeal carriage, invasiveness and disease incidence. The PPV is a 23-valent vaccine, which covers 23 commonly encountered serotypes. It is recommended to persons older than 65 years of age and aged ≥2 years at high-risk for pneumococcal diseases. The CPS is a T-cell-independent immunogen. It does not lead to isotype switching and induction of memory B-cell responses, leading to a temporary protection [ elderly . It has  elderly . The ove elderly . In aduleumoniae , is one eumoniae ,3; it iseumoniae ,4. It iseumoniae . The incnfection . The burnfection . Currentne (PCV) ,7. Both ne (PCV) ,9. S. pnotection . Capsulaotection ,12,13. Totection , resultiotection ,15 and rotection . The conotection . The intotection ,19. An eotection ,21 with S. pneumoniae is still urgent. The new vaccine should be able to induce more effective and durable immune responses that could potentially protect against all clinically relevant pneumococcal capsular types and cover some high-risk groups who may not respond well to the current vaccine, while keeping the cost low enough to be used in developing countries. The success of vaccines against other pathogens encourages the scientific community to develop a pneumococcal vaccine based on conserved protein antigens across all capsular types [S. pneumoniae vaccines [At the same time, some non-vaccine serotypes become prevalent in the face of the introduction of conjugate vaccines ,23,24. Aar types . Differevaccines ,27,28, avaccines ,30, antivaccines ,31,32,33vaccines .Salmonella and live lactic acid bacteria (LAB), mainly Lactococcus and Lactobacilli. Recombinant bacterial strains have several advantages. They have intrinsic adjuvant properties and can deliver antigens or DNA vectors with its native form using mucosal routes, which mimic the natural infection process to induce immune responses against the heterologous antigens in both mucosal and systemic sites. The productions of these kinds of vaccines are easier and less expensive than that of protein-based subunit vaccines. Several of the best-characterized candidate S. pneumoniae antigens, including pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal surface protein C (PspC), and pneumolysin (Ply), have been tested in various live vectors including attenuated pathogenic bacteria and nonpathogenic bacteria  can attach to, invade and colonize in deep effector lymphoid tissues after mucosal delivery and therefore remodel the host cells that they target as well as promote immunomodulatory effects to induce immune responses in locations where bacteria persist as well as at systemic sites [S. typhi vaccine vectors—χ9633, χ9639 and χ9640—delivering pneumococcal antigen PspA were safe and well-tolerated [S. pneumoniae strains. utations ,36. Furtic sites ,38,39. CS. typhimurium vectors were tested in mice  by intraperitoneal (i.p.) and oral routes [Salmonella vectors. Ply has activities of cytotoxic/cytolytic and complement activation to facilitate the growth and invasion of nfection . It is cnfection . Paton el routes . Both imS. pneumoniae CPS groups [S. pneumoniae [S. typhimurium vaccine vector delivers PsaA in two ways, one as a prokaryotic synthesized antigen [Salmonella strain χ9241 encoding full-length PsaA induced significantly high titers of anti-PsaA IgG in serum and IgA in vaginal washes, nasal washes, and lung homogenates [S. pneumoniae Tigr4 strain, Salmonella-PsaA vaccine reduced nasopharyngeal colonization by L82016 (type 6B CPS) and E134 (type 23 CPS) in two strains of mice, BALB/c (haplotype H2d) and C57BL/6 (haplotype H2b), independent of whether mice were immunized by the oral or intranasal (i.n.) route. However, immunization could not reduce lung colonization by pneumococcal strains A66.1 (type 3 CPS) and D39 (type 2 CPS). The Salmonella-PsaA vaccine conferred no protection against i.p. challenge with S. pneumoniae strain WU2 (type 3 CPS). The work also indicated that the full length of PsaA with its native conformation might be important to induce protective immunity, which is different from PspA, in which the α-helical segment is enough to induce protective immune responses [PsaA is a conserved surface protein present in all 90 S groups ,83,84. P antigen , another antigen . Salmoneogenates . Althougesponses . C-terminal 100 amino acids of the α-helical region [S. pneumoniae strains expressing different CPSes and serologically divergent PspAs [Salmonella vectors, was tested by different groups using either prokaryotic expression vectors or eukaryotic DNA vectors in mice  with its native secretion signal encoded on a high copy plasmid is delivered by a S. typhimurium SR-11 Δcya Δcrp strain [Salmonella-susceptible BALB/cJ mice (haplotype H2d) and Salmonella-resistant CBA/N xid mice (haplotype H2k) [Salmonella-PspA were protected against i.p. challenge with the virulent WU2 strain. The passive protection experiments showed that the immune serum can protect mice against intravenous (i.v.) (CBA/N mice) and i.p. challenge (BALB/c mice) with the WU2 strain. This work set the foundation for the following studies [In our group, Nayak p strain . This coype H2k) . Enzyme- studies ,50.et al. replaced the native secretion signal for the PspA Rx1 with the β-lactamase secretion signal [Salmonella SL1344 vaccine strain χ8501 (Δcrp-28 ΔasdA16), delivering this novel plasmid pYA3494, induced serum IgG and vaginal IgA against PspA and conferred protection against i.p. challenge with the WU2 strain. Notably, with only one-time immunization, the IgG response induced by χ8501(pYA3494) against PspA was higher than that against LPS or OMPs, both are indications of the immunogenic potential of the bacteria. This was also observed in a RASV strain with a regulated programmed lysis system vectoring the same bla SS-PspA fusion protein , including the N-terminal signal sequence of β-lactamase (bla SS), N- and C-terminal sequence of β-lactamase (bla SS+CT), ompA SS and phoA SS, fused with the α-helical domain of PspA Rx1 (encoding PspA Rx1 amino acids 3–285) on the immune responses [bla SS–PspA fusion yielded the largest amounts of secreted PspA than other signals. Mice immunized with this construction developed the highest serum IgG and vaginal IgA antibody levels, IL-4 and IFN-γ secretion. Immunized mice were protected against i.p. challenge with a virulent S. pneumoniae strain. Thus, the PspA production level in RASVs is important for the protection against S. pneumoniae challenge [Xin esponses . The reshallenge . Salmonella strains vectoring plasmid pYA4088 has been documented through a series of studies  and Th2-specific cytokines . Vaccination with χ9558 confers a greater degree of protection against S. pneumoniae challenge than that with χ8133  [S. pneumoniae challenge [Strain χ9558 is the representative of a new generation of RASVs displaying wild-type characteristics at the time of immunization and becoming attenuated after colonization of host tissues. It exhibits an improved safety profile in adult mice, with a reduced ability to cause meningitis when administered orally, i.n., or i.p. . It is te parent . In adul < 0.01) . Strain hallenge .bla SS-PspA-CT (PspA aa 3–285) was used to probe the protective immune mechanisms of RASVs via the oral route. Park et al. proved that the sIgA is important to RASV-PspA-induced protection against intratracheal (i.t.) challenge using pIgR−/− mice which lack the IgA secretion pathway [S. pneumoniae challenge. Influenza infection followed by pneumococcal infection can cause severe pneumonia and this secondary pneumococcal pneumonia is the most common cause of influenza-associated death. Seo et al. tested whether the vaccine against S. pneumoniae could reduce the disease burden caused by seasonal epidemic and pandemic influenza [S. pneumoniae and influenza, with a RASV against S. pneumoniae.Another plasmid pYA3802 with  pathway . Peyer’snfluenza . Mice vanfluenza . Thus, oS. pneumoniae and protects mice against pneumococcal challenge in carriage [et al. evaluated PspC from S. pneumoniae strain L82015 fused with different secretion signals as mentioned above [bla SS-PspC-CT fusions yielded the largest amounts of secreted PspC, induced the highest serum IgG and vaginal IgA titers, highest IL-4 and IFN-γ responses, and conferred the greatest protection against virulent S. pneumoniae i.p. challenge than other signal sequences fused to PspC. These results are consistent with the PspA results, which demonstrate that the antigen synthesis levels in live bacterial vectors are critical for induction of protective immune responses against S. pneumoniae. Using LAB as vectors delivering PsaA also confirmed this conclusion [PspC is another candidate surface antigen ,94. It pcarriage  and sepscarriage . Xin et ed above . The indnclusion . S. pneumoniae, multiple antigens are preferred to set blockages during the stages that S. pneumoniae attaches to, invades into and spreads in the host. Salmonella has the capacities to deliver multiple antigens with various approaches: (1) as fusion antigens on one plasmid delivered by one strain [To develop an effective vaccine against e strain ; (2) as e strain ; and (4)e strain . These aS. pneumoniae strains [S. pneumoniae strains [S. pneumoniae by i.p., i.v., or i.n. challenge [S. pneumoniae vaccine development. We first tested if a RASV strain delivering one vector encoding PspA fusions could induce protections against multiple  strains . PspA is strains . It is n strains ,97. We ehallenge . This fu+ vector to deliver the pspC gene, together with an Asd+ plasmid carrying the pspA gene to form a dual-plasmid system, which could deliver multiple antigens in a vaccine strain with Δalr ΔdadB and Δasd mutations [+ plasmids are compatible with Asd+ vectors in a single vaccine strain without comprising the synthesis of individual antigens. Both plasmids are stable over 50 generations of growth, suggesting that antigen synthesis and delivery in vivo are not compromised in this system [recF mutation was introduced into strains [Salmonella vaccine strain carrying both PspA and PspC by Asd+ and DadB+ vectors, respectively, induced higher serum and secretory antibody responses than the strain delivering a single antigen or a mixture of two vaccine strains each specifying one protective antigen and offered superior protection against i.p., i.v., or i.n. challenge with different serotypes of S. pneumoniae [+-Asd+ dual-plasmid system represents another important tool to develop multivalent live recombinant vaccines [We then evaluated the way to deliver multiple antigen genes in separate vectors in the case that a fusion construct of multiple protective antigens is not the optimal choice when a multivalent vaccine is desired. The major challenge to achieve this goal is that the recombinant vaccine strain should stably maintain two or more expression vectors simultaneously, each carrying a unique selectable marker. To facilitate this strategy, we used a DadButations . The Dads system . To furt strains . The Saleumoniae . The Dadvaccines .psaA and pspA have been shown to be effective in inducing antibody responses and Th1 immunity [et al. used Salmonella to deliver multi-antigen-encoding DNA vaccines encoding psaA and pspA genes [psaA and pspA genes. Salmonella delivering DNA vaccines encoding pspA or psaA, either alone or mixed together, significantly reduced S. pneumoniae colonization in nasal washes compared with control. Mice orally immunized with RASV carrying multi-antigen DNA vaccines significantly reduced nasal colonization by S. pneumoniae strain D39 compared to immunization with DNA vaccines administered intramuscularly (i.m.). These findings are related to the high level of sIgA in the nasal washer, as well as systemic IgG antibodies and a shift toward a Th1-mediated immune response [DNA vaccines encoding immunity , which aimmunity ,99,100. immunity . These ppA genes . They moresponse .Salmonella vaccines is that the DNA cannot effectively contact with the cytosol and then the nucleus of eukaryotic cells to initiate transcription and translation of encoded antigen genes. Besides those described above, other modifications could be used to increase the efficiency of DNA vectors delivered by Salmonella. Generally, there are two barriers for Salmonella delivering DNA vaccines into the cytosol. The first is that Salmonella resides in Salmonella containing vacuole (SCV) after entering the cell, which isolates Salmonella from other cytosolic components. This problem can be conquered by using a strain with the sifA mutation [sifA disrupts the vacuoles [Salmonella. This can be conquered by using a regulated delayed lysis in vivo strategy [One of the main problems with DNA vaccines delivered by live mutation . SifA isvacuoles . The secstrategy . This ststrategy . S. typhi vaccines for human use is slow. Currently, a clinical trial is still the best measurement of safety and effectiveness of S. typhi vaccines or vaccine vectors. Our intensive work carried out in mice lead to the development of a S. typhimurium strain χ9558 with a balance between safety and immunogenicity in adult, neonatal and infant mice [S. typhi vaccine vectors, χ9633, χ9639, χ9640, with essentially the same genotype as χ9558 carrying plasmid pYA4088 encoding the α-helical fragment of PspA Rx1 (aa 3–285) [Salmonella vaccines is, at least in part, on its RpoS status. All three S. typhi vaccine strains are similar to the licensed live attenuated typhoid vaccine Ty21a in their abilities to survive in human blood and human monocytes. They are more sensitive to complement and less able to survive and persist in sewage and surface water than their wild-type counterparts [Salmonella antigens. The percentages of protection against S. pneumoniae challenge in adult mice immunized with these vectors are between 50 and 81.3% [7 to 1010 CFU to further evaluate the safety and immunogenicity and determine which of the three S. typhi vectors has the optimal safety and immunogenicity profile in human hosts [S. typhi and PspA likely due to previous Salmonella infection and pneumococcal vaccination, possible over-attenuation or limited in vivo slow growth of the attenuated S. typhi strains. In this last regard, the use of the regulated delayed synthesis of PspA in vivo might have been due to too much repression and insufficient cell divisions of the vaccine strains to adequately reduce repressor concentration by cell division. Based on the trial results, the vaccine strains have been further modified to increase protective antigen production and delivery to increase immune responses. An improved version of S. typhi based on the most promising vaccine strain χ9640 carrying vectors encoding multiple protective pneumococcal antigens is being developed and evaluated. Due to the lack of an animal model, progress to develop safe ant mice ,56,91,92a 3–285) , but wita 3–285) ,66. The terparts . Adult, nd 81.3% ,66. In tnd 81.3% ,66. ThesS. typhi is the choice for oral human vaccines because it can effectively invade mucosal tissues and enter systemic sites, leading to strong mucosal, humoral and cellular immune responses. S. typhimurium only causes self-limited gastroenteritis in human. It is less capable to invade beyond the gut mucosa in healthy humans and less able to stimulate long-lasting immunity [S. typhi vaccine strains, infection of mice with S. typhimurium is used as an experimental model because S. typhimurium infection in mice results in typhoid-like diseases in mice, which likens S. typhi infection in human. Although Salmonella shows promise as a vaccine vector and has been extensively tested, there is still no licensed RASV. In addition to the issue of immunogenicity, the key concern associated with RASVs is safety, especially in newborns/neonates, the elderly and those who are immunocompromised or have chronic diseases. Clinical results demonstrated that our RASVs χ9633, χ9639 and χ9640 are safe and non-shedding, but less immunogenic [S. typhimurium as a model. Our S. typhimurium vaccine vector χ9558 carrying plasmid pYA4088 induced significant anti-PspA IgG/IgA antibody titers in mice, S. typhi vaccine strains χ9633, χ9639 and χ9640 with the same genotype carrying the same plasmid did not induce significant anti-PspA IgG/IgA responses in the clinical trial. The next step is to increase their immunogenicity while maintaining safety. Attaining the desired balance between safety and immunogenicity is difficult, especially for S. typhi vaccines, which lack a relevant animal model. To develop a S. typhi vaccine, evaluation of S. typhimurium strains of similar genotype and phenotype in mice is used as a close mimic of S. typhi in humans. Considering the differences between the human and mouse mucosal lymphoid system [. However, they still have problems with variations in ability of S. typhi to attach to, invade into, survive intracellularly and distribute into internal effector lymphoid tissues. These disadvantages and the high cost of these humanized mice still limit their current use. Further research and improvement of these humanized mouse models should ultimately aid in developing a safe and effective S. typhi vaccine vectors for humans [Human-restricted immunity . Thus, iimmunity . To evald system ,107, sevd system ,110,111.r humans .S. typhi RASVs used adult mice, but a few studies have adopted using newborns and neonates in developing pneumococcal vaccines [S. pneumoniae challenge [S. typhimurium vaccine and enhance levels of mucosal IgA, IFN-γ, and IL-4 in mice immunized with S. typhi vaccines [S. pneumoniae vaccines. We may need age-specific RASVs for different age groups.Currently, the mouse is still the most cost-effective model for testing safety and efficacy of RASVs. Most studies with vaccines ,66,91 In vitro, L. rhamnosus can inhibit S. pneumoniae adherence to human epithelial cells [S. pneumoniae in humans [L. lactis in mice can improve clearance of pathogens from the lungs, reduce lung injuries, and increase survival of mice against S. pneumoniae infection [+ cells in the intestine and bronchus, and increase BAL anti-pneumococcal IgA and IgG levels [L. fermentum in mice can increase protective responses against S. pneumoniae challenge by stimulation of neutrophil activity or by the increase of the number of activated macrophages and lymphocyte populations in the tracheal lamina propria [L. lactis improves local and systemic immune responses against S. pneumoniae with reduced nasal colonization, increased clearance rate of S. pneumoniae from lungs, reduced dissemination of pneumococci into blood and reduced damage to respiratory tissues, which is also related to the up-regulation of the innate and adaptive immune responses in both local and systemic compartments as well as different cytokine responses [L. casei [S. pneumoniae [Live LAB have been shown to be effective adjuvants to improve the immune responses against respiratory pathogens ,136. In al cells . Ingestin humans . Oral adnfection ,140. Thenfection . These rG levels . Nasal a propria ,143. Nasesponses . These rL. casei . Howevereumoniae ,145,146.L. lactis NZ9000 or its parent MG1363, have been tested against S. pneumoniae challenge in animal models. The strain NZ9000 is derived from the strain MG1363 with the nisRK genes integrated into the pepN gene, which facilitate the use of the NIsin-Controlled gene Expression system-NICE [S. pneumoniae: recombinant lactic acid bacterial vectors and non-genetically modified Gram-positive enhancer matrix (GEM) particles. We will focus on the live vaccine strategy. The GEM approach was discussed elsewhere [Several pneumococcal candidate antigens, PsaA, PspA, PspC and PppA delivered by LAB, most in tem-NICE ,148. Twolsewhere ,151,152.S. pneumoniae vaccines. One is the lactococcal promoter P1 and another is the nisin-regulated promoter [psaA, pspA and pspC [L. lactis genome [To induce immune responses, especially antibody responses, higher antigen production is preferred. Thus strong promoters are adopted. Although several promoters were used in LAB , two propromoter ,71,153. and pspC ,71,153, s genome . Howevers genome . To solvnis can be efficiently controlled by the extracellular concentration of the antimicrobial peptide nisin through the two-component regulatory system, sensor NisK and regulator NisR [L. lactis F17847, with the NICE system, the induction of antigen synthesis with nisin before immunization is necessary. Other strains, like L. lactis NZ9700, can produce nisin to omit this process. However, it also means that the NICE system is converted into a non-regulated system. Basically, the NICE system is a system that regulates protein synthesis in vitro, not in vivo. The nisin-regulated promoter system has several advantages. The transcription of the promoter Ptor NisR ,155,156,tor NisR ,157. Fortor NisR . As an aL. lactis. The secretion of Usp45 is through the Sec pathway. Adding negative charge peptides at the N-terminal part of the mature moiety will improve the translocation efficiency across the cytoplasmic membrane [Salmonella, in which periplasmic secreted antigen induced higher antibody responses than cytoplasmic antigen [Another way to reduce the metabolic burden on the LAB vector is to secret the protein into the periplasm, onto the cell wall or into the supernatant. The secreted protein can directly interact with the environment. Several protein secretion systems in LAB have been discussed ,134,158.membrane ,160. Thumembrane ,153. Howmembrane ,162,163. antigen . AlthougS. pneumoniae vaccines. Recombinant LAB delivering pneumococcal antigens is mainly by the i.n. route. LAB strains were used to deliver PsaA, PspA and PspC by nasal immunization ,69,153 aLactobacillus synthesizing PsaA developed systemic anti-PsaA IgG and IgA responses and displayed reduced pneumococcal colonization upon nasal challenge [Lactobacillus from 20 to 250 ng/109 cells [L. plantarum and L. helveticus induce significant IgA responses in nasal and bronchial washes and IgG in serum as well as reduced nasal colonization of S. pneumoniae 6B compared with the saline control group. However, when compared with strains carrying the control vector, only the recombinant L. helveticus led to a significant reduction of pneumococcal nasal colonization. Although L. casei does not generate significant antibody responses, it results in reduced colonization compared with the saline group, but not the vector control. These results reflect that LAB strains have different adjuvant properties. The three LAB strains synthesize similar amounts of PsaA (150–250 ng/109 cells). They persist in the mice nasopharynx after inoculation for three days except L. casei [Mice intranasally immunized with some species of hallenge . The imm09 cells . L. planL. casei . Short pL. casei . L. casei and L. lactis were used to deliver PspAs. L. casei delivered PspAs from clades 1 and 5 under the control of the constitutive P1 promoter. The PspA synthesized retains in the cytosol [L. casei-PspA1/5 develop significant anti-PspA IgG, but no IgA in nasal washes, saliva or vaginal washes [L. casei seems not to display adequate adjuvant activity [S. pneumoniae. Mice immunized with L. casei-PspA1 show increased survival times when compared to mice immunized with saline against lethal i.p. pneumococcal challenge [L. casei-PspA1 bacteria can be recovered up to five days after the i.n. inoculation with 109 CFU on two consecutive days. The presence of PspA antigen does not affect the ability of strain colonization in the nasal pharynx [L. casei delivering PspA5 conferred protection against i.n. pneumococcal challenges in mice. This was accompanied by the increased secretion of IFN-γ by lung cells against invasive pneumococcal challenge [ cytosol ,70. Micel washes ,70. Prevl washes ,165, thuactivity . The anthallenge . Howeverhallenge . L. case pharynx . AnotherL. lactis with intracellularly produced PspA using the NICE system induced not only serum anti-PspA IgG, but also lung lavage anti-PspA IgA [L. lactis-PspA significantly protected mice against i.p. challenge with the S. pneumoniae TIGR4 strain than protein administered intranasally or control groups did. The protection induced by L. lactis-PspA is similar to that induced by PspA/Alum administered by the subcutaneous (s.c) route. This was attributed to the Th1-mediated immune responses induced. In an intranasal challenge model, L. lactis-PspA afforded the highest protection among the levels elicited with purified PspA administrated i.n. or s.c. with adjuvant or in control groups [L. lactis may contribute to non-specific host immunity.Intranasal immunization with PspA IgA . This rel groups . About 2et al. reported that L. casei-PspC (from CPS type 6B) without an SS cannot confer significant protection against i.n. pneumococcal challenge although it induces IFN-γ secretion in lung cells and IL-17 secretion in both lung and spleen cells [et al. tested L. casei-PspC with or without an SS using different immunization protocols, intranasal, sublingual and primer-boosting with PspC protein. However, none of these protocols induced significant levels of anti-PspC antibodies in vaginal or nasal washes and serum before challenge [L. casei-PspC without a SS significantly reduced pneumococcal colonization by strain 0603 with an increase of anti-PspC IgA in the nasopharynx five days after challenge [L. casei carrying cell-wall-associated PspC only marginally reduced pneumococcal colonization after challenge. Thus, the reduced colonization of S. pneumoniae may be attributed to the non-specific adjuvanticity of L. casei. These results show that protection is only achieved by using a PspC with high identity at the N-terminal region to the PspC expressed by the L. casei vaccine strain. Considering the polymorphism of PspC [Ferreira en cells . This mahallenge . Despitehallenge . L. case of PspC , using d of PspC . L. lactis NZ9000 on the cell surface, was tested as live or inactive vaccines using intranasal and oral routes in adult and young mice [L. lactis-PppA induced anti-PppA IgM, IgG and IgA responses in serum and bronchoalveolar lavage fluid in both adult and young mice. The responses are significantly higher in young mice than in adult mice. The challenge results showed that intranasal immunization with L. lactis-PppA could confer protection against homologous S. pneumoniae i.p. challenge by either active immunization or passively by antibody from immunized mice and increased resistance to respiratory infection with different pneumococcal serotypes  in young and adult mice [L. lactis-PppA provided cross-protective immunity against four CPS types of pneumococcal strains with reduced lung bacterial counts [L. lactis found in the serum or the BAL fluid in adult and young mice immunized with the recombinant strain. Thus, the host immune responses are directed against the protein expressed by L. lactis, not the vector [L. lactis-PppA strain is more effective [An antigenically conserved antigen, PppA , deliverung mice ,73,74,75ult mice ,73,74,75l counts . There ae vector . These rffective . eps natural promoter to express the eps genes or the nisin-induced promoter to express the regulatory genes, respectively [L. lactis expressing 0.5 μg of type 3 CPS or 0.5 μg of purified type 3 CPS from pneumococcus elicited similar titers of T-cell-independent anti-CPS IgM and IgG antibodies in the serum [L. lactis is a potential host for capsular synthesis. However, there is no animal protection study with CPS delivered by LAB vectors. LAB were also used to deliver type 3 or type 14 CPS with the ectively ,77. The ectively  or in thhe serum . The LABhe serum . Thus, LS. pneumoniae antigens used i.n. immunization (8) of LAB for consecutive five days is preferred to induce IgA responses [Though LAB are normally ingested orally, most work with LAB delivering nization . One of esponses ,141. Mosesponses ,170,171,esponses . Also, fSalmonella by i.n. route results in brain colonization if the bacteria is not fully attenuated [Another issue related to the LAB vaccine is the safety concerns of i.n. immunization. I.n. immunization of mice with recombinant LAB induces excellent immune responses to the expressed antigens. Since the cribriform plate is a thin, well-hidden bone in the nasal cavity with numerous perforations for allowing passage of the olfactory nerves to the brain, there exists a potential route for bacteria to enter the cranial cavity if administered by i.n. route. Immunization with tenuated . The GRAS. pneumoniae vaccines is the use of antibiotic-resistance markers, which are considered unacceptable in live vaccines due to the potential for antibiotics in the final product and the possible contamination of the environment with recombinant drug-resistant bacterial strains. The regulatory agencies also prohibit the usage of antibiotics in vaccine formula. Two antibiotics are commonly used in the LAB vectors. The first one is erythromycin. Erythromycin can inhibit the protein synthesis by binding the 50s subunit of the bacterial 70s rRNA complex. Most plasmids used in LAB vaccines have an erythromycin-resistance selection marker. This antibiotic is necessary to select the recombinant plasmid. LAB strains with recombinant plasmid are grown with erythromycin prior to immunization to maintain the plasmid. Adding the antibiotics not only increases the costs of the final product, but also raises the concern about the plasmid stability. The most used pTREX vectors have poor segregational stability in the absence of antibiotic selection [in vivo and lead to compromised immune responses. To conquer this problem, the use of the balanced-lethal strategy could be attempted with LAB vaccines. Another is the nisin. Nisin is a polycyclic lantibiotic produced by L. lactis to eliminate other competing Gram-positive bacteria. It is commonly used as a safe food preservative against bacteria, yeast, and molds. Nisin can bind to lipid, dissipate the membrane potential, induce efflux of cytoplasmic components and inhibit bacterial cell growth [Listeria spp. at a frequency as high as 10–4 [Another disadvantage with the LAB-election . LAB coul growth . It is ul growth , which a as 10–4 . Nisin c as 10–4 . Therefo as 10–4 ,179,180.Salmonella need to be attenuated to achieve a balance between safety and immunogenicity for vaccine application. It is not necessary to generate and evaluate mutations in LAB due to their GRAS feature. However, LAB have different abilities to modulate the immune system, the careful selection of LAB should be noted as a key factor that influences the results. Different LAB strains induce distinct cytokine profiles and exert different effects on the immune system [L. casei CRI 431, L. lactis NZ9000 and L. rhamnosus CRL1505 have proved to be able to increase the resistance of mice to challenge with respiratory pathogens. A human study showed that L. rhamnosus GG has different immune modulation functions [S. typhi vaccine administrated with L. rhamnosus GG induced higher numbers of IgA-secreting cells, while with L. lactis induced higher numbers of CR3 receptor expression on neutrophils [L. lactis-PspA can induce effective IgA responses, but L. casei-PspA is poor for induction of an IgA response [L. lactis, L. casei, L. plantarum and L. helveticus , a live attenuated strain of  infants . Besides infants , it has  infants . This lige model . The anthallenge , againstPS types . Thus, teumoniae . et al. used replication-defective recombinant adenoviruses Ad5 (rAds) to deliver PspA, PsaA and PdB, either individual or combined [S. pneumoniae. rAd/PdB+rAd/PspA+ rAd/PsaA is most effective in reducing the bacterial load in the lung after challenge. There are a few reports using viral vectors to deliver pneumococcal antigens. Arévalo combined . rAds cacombined . The rAdcombined . The resS. pneumoniae vaccine research [et al. reported that candidate antigens, such as PsaA, Stkp, PcsB, show different requirements for stability when combined with different adjuvants and excipients [E. coli was developed [in vivo but preserve the bacteria at 2–8 °C for at least 12 months with only 0.4 log10 loss of viability during storage. These results provide a practical option for formulating ETEC vaccines or other live bacterial vaccines for oral immunization and help to facilitate delivery of lifesaving vaccines, particularly in low-resource settings [Protein-based vaccines are the future for research . Howevercipients . PsaA neeveloped . The tabsettings . Our expsettings . WhetherS. pneumoniae causing disease involves multiple steps, including attaching to, invading into and spreading in the host [S. pneumoniae also have over 90 serotypes with different pathogenic attributes. The vaccine should protect against infections by most of the serotypes. The ideal vaccine should include multiple antigens to stop multiple steps of infection and confer protection against multiple serotypes. In the future, work should focus on delivering multiple antigens and test protection with multiple challenge models against multiple serotypes. Currently, the challenge studies in evaluating recombinant live vaccine vectors used serotypes 2, 3, 4, 6B, 14, and 23F (S. pneumoniae strains in multiple families with regard to the PspA and PspC antigens is therefore important. The inclusion of conserved antigens such as PsaA and Ply and the conserved proline-rich domains of PspA and PspC should further augment the protective efficacy of these vectored vaccines [the host . An effe and 23F , which dvaccines ,63.S. pneumoniae. These improvements enhance safety, tolerability and effectiveness in inducing protective immunity. Three S. typhi vaccines based on these technologies have been tested in a phase I clinical trial. As for LAB based vaccines, there is still room for improvement. In the future, combining the benefits of LAB and Salmonella will be possible. With the advancement of knowledge about bacteria—not only Salmonella and LAB, but also other bacteria interacting with hosts—and by using new technologies, we should finally be able to develop safe, efficient, and relatively inexpensive needle-free vaccines against S. pneumoniae, as well as other pathogens. Much progress has been made in improving the technologies to design, construct and evaluate live bacterial vectored vaccines against


EYS are likely pathogenic mutations in two arRP families and eight sporadic patients. Specifically, we found a novel pair of compound heterozygous mutations and a novel homozygous mutation in two separate arRP families, and found two novel heterozygous mutations in two sporadic RP patients, whereas we found six novel homozygous mutations in six sporadic RP patients. Of these, one was a frameshift mutation, two were stop-gain mutations, one was a splicing mutation, and the others were missense mutations. In conclusion, our findings expand the spectrum of EYS mutations in RP in the Indian population and provide further support for the role of EYS in the pathogenesis and clinical diagnosis of RP.Retinitis pigmentosa (RP) is a rare heterogeneous genetic retinal dystrophy disease, and despite years of research, known genetic mutations can explain only approximately 60% of RP cases. We sought to identify the underlying genetic mutations in a cohort of fourteen Indian autosomal recessive retinitis pigmentosa (arRP) families and 100 Indian sporadic RP cases. Whole-exome sequencing (WES) was performed on the probands of the arRP families and sporadic RP patients, and direct Sanger sequencing was used to confirm the causal mutations identified by WES. We found that the mutations of  However, these genes account for only approximately 60% of RP casesRetinitis pigmentosa RP; OMIM 226800) is a highly heterogeneous genetic disease characterized by progressive visual loss caused by the impairment of retinal photoreceptors26800 is EYS gene  corresponds to the RP25 locus and was identified as the gene causing autosomal recessive retinitis pigmentosa (arRP) in 2008, and it is mainly expressed in the retinaEYS gene encodes a homologue of the Drosophila eye spacemaker (SPAM) protein and is essential for the development and morphology of photoreceptorsEYS for RP patients, and the types of mutations include missense mutations, nonsense mutations, insertions, deletions and splice site mutations26789The EYS gene in two consanguineous Indian families and eight sporadic Indian RP patients. Our findings expand the mutation spectrum of EYS within the Indian population and demonstrate that WES by NGS is a powerful tool for the genetic diagnosis of RP.However, there has been limited success when using traditional approaches to screen potential genes for RP because many techniques for positional cloning and gene identification are relatively time consuming, expensive and inefficient. Recently, whole-exome sequencing (WES) by next-generation sequencing (NGS) has become an efficient method for identifying genetic variants at the whole-genome level. In several studies, NGS has provided a promising alternative approach for the molecular diagnosis and genetic identification of RP11All study protocols were approved by the Ethics Review Board of Aravind Medical Research Foundation of India and the Hospital of the University of Electronic Science and Technology of China and the Sichuan Provincial People’s Hospital, and all experiments were performed in accordance with the approved protocols. Fourteen families with arRP, 100 patients with sporadic RP and 1000 control individuals with no history of retinal diseases were recruited in this study. Samples were collected from the Aravind Eye Hospital of India. Written informed consent was obtained from all individuals who participated in this study or from their legal guardians in the case of minors. Venous blood samples were obtained from all subjects in EDTA vacutainers.Total genomic DNA was isolated with DNA extraction kits according to the manufacturer’s instructions  and stored at −20 °C for later use. The integrity of the DNA was assessed through 1% agarose gel electrophoresis.http://genome.ucsc.edu/) using the Burrows-Wheeler Aligner (BWA) program v.0.5.9-r16 (http://bio-bwa.sourceforge.net/)http://samtools.sourceforge.net/) using the ‘mpileup’ command. The variants that had depths less than 10 and those located outside of the exome-capture regions were filtered out. For all of the samples, we used individual base-calling algorithms.DNA samples from the probands of arRP families and 100 sporadic RP patients were subjected to WES at Axeq Technology Inc., Seoul, Korea. In brief, the workflow of WES was as follows. First, the genomic DNA samples were fragmented into 150–200 bp fragments and then ligated to paired-end adaptors. Exome enrichment was performed according to the manufacturer’s protocol with the Agilent SureSelect Human All Exon 50 MB kit V5 , which covers 20,965 genes and 334,378 exons in the Consensus Coding Sequence Region database. The captured libraries were subjected to a quality assessment with an Axeq 2100 Bioanalyzer and sequenced on an Illumina HiSeq 2000 sequencer. The raw image files were processed by Illumina base calling software v.1.7 for base calling with default parameters, and the sequences of each individual were generated as 90-bp paired-end reads. High-quality sequencing reads of each sample were aligned to the reference human genome hg19 UCSC assembly (http://sourceforge.net/projects/snpeff/files/databases/v4_1/).SnpEff v4.1 software was used for variants annotation (http://www.ncbi.nlm.nih.gov/CCDS/ccdsBrowse.cgi), RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq/), Ensembl (http://www.ensembl.org), and Encode (http://genome.ucsc.edu/ENCODE). Exclusion steps were performed to identify the candidate variants. Variants within intergenic, intronic and UTR regions and synonymous mutations were excluded, and variants in dbSNP138 (http://www.ncbi.nlm.nih.gov/projects/SNP/), the 1000 Genome project (ftp://ftp.1000 genomes.ebi.ac.uk/vol1/ftp), the YH database (http://yh.genomics.org.cn/), the HapMap Project (ftp://ftp.ncbi.nlm.nih.gov/hapmap) and our in-house database generated from 1600 samples sequenced by WES were also excluded. Moreover, potentially damaging effects of the variants on protein structure/function were predicted by SIFT (http://sift.bii.astar.edu.sg/) and Ployphen2 (http://genetics.bwh.havard.edu/pph2).The detected variants were filtered and annotated based on four databases: NCBI CCDS (EYS gene reference sequence (NC_000006.12) to confirm the candidate nucleotide variants.Sanger sequencing was performed to confirm the variants identified by WES. Primers flanking the candidate loci were designed using Primer 5.0 and synthesized by Invitrogen, Shanghai, China. The primers used for Sanger sequencing are shown in The detailed clinical data for two arRP families and eight sporadic RP patients are shown in EYS gene were identified in two arRP families and eight sporadic RP patients. The detailed WES results for these patients are illustrated in EYS gene (NM_001142800). In family ARRP-206, we found a homozygous single-nucleotide mutation (c.1871G>A (p.S624L)) )  in ARRP-.S624L))  and 3. TEYS, such as the Laminin G (LamG), EGF, EGF-like calcium-binding (EGF-CA), and EGF-like domains  and our 1000 control results, homozygous mutations or compound heterozygous mutations were not detected in the normal control population.Additional disease-causing mutations of other known RP genes or previously identified mutations of EYS protein function. Most mutations are located at the protein functional domain LamG or EGF/EGF-like/EGF-CA, which may affect the function of the protein  or EGF-like domains at the N-terminus and five LamG domains at the C-terminus and is highly expressed in retinal photoreceptorsEYS in Drosophila named Spacemaker (spam) is involved in luminal space formation and plays an essential role in the formation of matrix-filled interrhabdomeral space in Drosophila1516EYS function in the retina are essential for deep understanding of the pathological mechanism of retinal degeneration.EYS is a major causative gene for RP. Thus far, the mutations identified in EYS include p.D904Qfs*17, p.S754Afs*6, p.T657Afs*5, p.W2640*, p.E1836* and others59EYS protein, which reveals that the C-terminus of EYS is essential for its function in the retina. In this study, we found a pair of compound heterozygous mutations, c.8422G>A (p.A2808T) and c.7868G>A (p.G2623E), in EYS in family ARRP-49. Both mutations affect a conserved amino acid residue. The mutation p.A2808T in exon 43 is a single nucleotide polymorphism (rs111991705) with an allele frequency of 0.009826. This mutation results in the replacement of a small hydrophobic amino acid  by a polar residue (threonine). Previously, Audo, I., et al. reported the same heterozygous mutation c.8422G>T in a French RP patienthttp://www.uniprot.org/uniprot/Q5T1H1), which may affect the formation of disulphide bonds, thereby impairing protein function. These two mutations together may account for the incidence of RP in family ARRP-49. The novel homozygous mutation c.1871G>A (p.S624L) in EYS is likely the causative mutation for family ARRP-206. This missense mutation leads to the replacement of a small-sized polar residue, serine, by a hydrophobic lysine. However, this mutation is not highly evolutionarily conserved, and further studies are warranted to ascertain the impact of this mutation on EYS functions.The majority of RP cases show the sporadic form19EYS. These nonsense mutations could cause the degradation of EYS mRNA via nonsense-mediated mRNA decay (NMD)et al., demonstrated that mutation types were related to the severity of the RP symptomsIn sporadic patients RP:S-22 and RP:S-48, we found two novel homozygous stop-gain mutations, c.8388C>A (p.Y2796X) and c.3024C>A (p.C1008X), respectively, and these mutations resulted in the generation of truncated proteins in the fourth LamG domain at the C-terminal and the fifth EGF-CA domain at the N-terminal, respectively. The truncated proteins likely impair the function of EYS and seven novel homozygous mutations for RP in the Indian population. Our study not only expands the spectrum of EYS mutations for arRP in the Indian population but also shows that WES can be an effective tool for identifying causative mutations in RP patients and diagnosing genetic diseases.In summary, we identified three novel compound heterozygous mutations in How to cite this article: Di, Y. et al. Whole-exome Sequencing Analysis Identifies Mutations in the EYS Gene in Retinitis Pigmentosa in the Indian Population. Sci. Rep.6, 19432; doi: 10.1038/srep19432 (2016).


PTPRQ are associated with deafness in humans due to defects of stereocilia in hair cells. Using whole exome sequencing, we identified responsible gene of family 1572 with autosomal recessively non-syndromic hearing loss (ARNSHL). We also used DNA from 74 familial patients with ARNSHL and 656 ethnically matched control chromosomes to perform extended variant analysis. We identified two novel compound heterozygous missense mutations, c. 3125 A>G p.D1042G  and c.5981 A>G p.E1994G , in the PTPRQ gene, as the cause of recessively inherited sensorineural hearing loss in family 1572. Both variants co-segregated with hearing loss phenotype in family 1572, but were absent in 74 familial patients. Heterozygosity for c. 3125 A>G was identified in two samples from unaffected Chinese individuals (656 chromosomes). Therefore, the hearing loss in this family was caused by two novel compound heterozygous mutations in PTPRQ.Mutations in  Autosomal recessive non-syndromic hearing loss (ARNSHL) accounts for about 80% of hereditary hearing loss , 2. To dGiven the genetic heterogeneity of ARNSHL, the deafness genes encode a large variety of proteins with different functions, including gene regulation, fluid homeostasis, synaptic transmission, and hair cell bundle morphology and development , 4. DefeTPRN, GPSM2, CEACAM16, SMPX, HSD17B4, HARS2, MASP1, DNMT1, TSPEAR, CRXCR2, etc [Researchers have recently identified more than 10 non-syndromic deafness genes and several dozens of novel causative mutations using targeted genomic enrichment and Whole Exome Sequencing (WES), including CR2, etc –14.PTPRQ is located at the DFNB84 locus on chromosome 12q21.2 and consists of 45 exons comprising 234,677 base pairs (bp). The protein encoded by this gene, PTPRQ, is a protein tyrosine phosphatase receptor type Q composed of 2,299 amino acids, and plays an important role in the formation of stereocilia in hair cells of the inner ear. Loss of Ptprq leads to shaft-connector malformation of hair bundle in the mammalian cochlea [ cochlea . It has  cochlea .PTPRQ have been shown to be associated with ARNSHL in three families from Dutch, Moroccan and Palestinian descent  and Sanger validation. These procedures identified two compound heterozygous disease-segregating mutations, c. 3125 A>G (p.D1042G) and c.5981 A>G (p.E1994G), within the PTPRQ gene. In addition, DNA samples from 74 patients from ARNSHL families and 328 unaffected individuals (656 chromosomes) were also analyzed.Here, we report a family with two siblings affected by congenital sensorineural hearing loss. Mutations within the GJB2 and SLC26A4 genes had been excluded previously. Fully informed written consent was obtained from all subjects or, in the case of children, from their guardians. The study was approved by the Chinese PLA General Hospital Research Ethics Committee. Medical histories, otoscopy, physical examination, immittance testing and pure tone audiometric examination of family 1572 members were performed as described in detail previously [Family 1572 is a two-generation Chinese family with autosomal recessive prelingual non-syndromic sensorineural hearing loss. To screen for candidate mutations, 328 ethnically matched controls (656 chromosomes) and 74 affected DNA samples from the Department of Otolaryngology, Head and Neck Surgery, Chinese PLA General Hospital were analyzed. The 74 affected patients originated from 74 families presenting with ARNSHL, and in whom mutations within eviously . The parAll genomic DNA was extracted from peripheral blood using a blood DNA extraction kit according to the manufacturer’s instructions .http://www.ncbi.nlm.nih.gov/sra website; accession number SRP050281).Whole-exome capture and sequencing have been described in detail previously . In brieRead mapping, variant detection, filtering, and annotation have been described in detail previously . AccordiAfter filtering against multiple databases, Sanger sequencing was used to determine whether any of the potential novel mutations within known causative genes of ARNSHL co-segregated with the disease phenotype in this family. Direct PCR products were sequenced as described in detail previously .PTPRQ GenBank sequence (NM_001145026.1) using the Genetool software.Mutation segregation was performed in all family members. Additionally, 328 negative samples and 74 familial patients were also screened for the mutations by direct sequencing. Genotyping for c. 3125 A>G and c.5981 A>G was performed by PCR (primer sequences are available upon request) and detected by bidirectional sequencing of the amplified fragments using an automated DNA sequencer . Nucleotide alteration(s) were identified by sequence alignment with the Homo sapiens), XP_001085761.2 (Macaca mulatta), XP_001151129.2 (Pan troglodytes), NP_001179484.1 (Bos taurus), XP_539698.3 (Canis lupus), NP_001074901.1 (Mus musculus), NP_075214.1 (Rattus norvegicus), XP_001235338.2 , and XP_683017.4 (Danio rerio).Multiple sequence alignment was performed according to a Homologene program with default settings and the following sequences: NP_001138498.1 (http://swissmodel.expasy.org/workspace/). This study used the automatic modeling approach to apply the complete protein sequence of human PTPRQ, including its 2,299 amino acids and its mutation, which are available in the NCBI GenBank (NP_001138498.1) in FASTA format. Data obtained by the homology models were visualized using Swiss-PdbViewer 4.1.Three-dimensional (3D) modeling of the human wild-type, p.D1042G mutation and p.E1994G mutation were performed using SWISS-MODEL, an automated homology modeling program  and two unaffected parents . AudiogrWES was applied to the two affected siblings and two unaffected parents: I:1, I:2, II:1, and II:2. The reads were aligned with the human genome reference sequence. On average, 6.30 billion bases of sequence were generated with mean target region depths of 73.6. Approximately 99.1%  of the targeted bases were covered sufficiently to pass our thresholds for calling single nucleotide polymorphisms (SNPs) and short insertions or deletions (indels). The average rates of nucleotide mismatch were 0.34%. After identification of variants, only non-synonymous variants (NS), splice acceptor and donor site mutations (SS), and frameshift coding indels were used for further analysis; these are more likely to be pathogenic than others, especially those in homozygous or compound heterozygous modes .http://www.ncbi.nlm.nih.gov/projects/SNP/), HapMap project (ftp://ftp.ncbi.nlm.nih.gov/hapmap), 1000 Genome Project (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp), and YH databases (http://yh.genomics.org.cn/). Potential candidate variants were identified by selecting the homozygous or compound heterozygous variants found in both affected siblings while unaffected parents contributed equally. Under the autosomal recessive mode of inheritance, 22 genes with compound heterozygous variants and two genes with homozygous variants were identified. Next, these variants were compared to reported non-syndromic hereditary hearing loss genes, then two mutations (c.3125 A>G and c.5981 A>G) were found in a previously reported deafness-related gene, PTPRQ.On average, 95,337 SNPs in the coding regions and 2,521 variants in introns were identified that may affect splicing (within 10 bp of the intron/exon junction) for each sample . Next, thttp://sift.bii.a-star.edu.sg/) and Polyphen (http://genetics.bwh.harvard.edu/pph2/), which predicted them to be tolerated. The c. 3125 A>G substitution occurs in exon 20 of PTPRQ and results in a single amino acid substitution  within the FNIII 11 domain. The c.5981 A>G substitution occurs in exon 37 of PTPRQ and results in a single amino acid substitution  within the topological domain. Both mutations affect the conserved residues  in two individuals among the 328 ethnically matched controls (656 chromosomes), and c.5981 A>G was absent in 656 negative chromosomes. These two mutations were absent in 74 familial patients as well as the dbSNP135, HapMap project, 1000 Genome Project and YH databases. These observations suggest that the p.D1042G and p.E1994G mutations are likely to have a detrimental effect on the protein.The D1042G molecular model covered the target sequence of PTPRQ , and was constructed based on the crystal structure of the axon guidance receptor (PDB ID: 4hljA). Sequence identity between the target and template was 25%. Using Swiss-Pdb Viewer 4.1, the mutation was predicted to perturb the amino acid side chain due to the substitution of aspartic acid to glycine at position 1,042. The mutant residue is also smaller than the wild-type residue .The E1994G molecular model covered the target sequence of PTPRQ , and was constructed based on the crystal structure of the catalytic domain of PTPRQ (PDB ID: 4ikcA). Sequence identity between the target and template was 97.13%, which is considerably higher than the average (25%). Using Swiss-Pdb Viewer 4.1, the mutation was predicted to perturb the amino acid side chain due to the substitution of glutamic acid to glycine at position 1,994 . The wilPTPRQ mutations are the cause of ARNSHL in this family.These data, together with the clinical presentation of the two affected siblings, clearly indicate that PTPRQ gene. PTPRQ mutations are rare causes of recessive deafness as there are only two reports worldwide. This is the first study to identify PTPRQ as the ARNSHL-associated gene in a Chinese population.We report here the use of WES approach in a small nonconsanguineous family including 2 affected individuals with ARNSHL. This approach led us to the identification of two novel compound heterozygous mutations in PTPRQ, a receptor-like phosphatase, have shown to be active against phosphatidylinositol phosphates which is involved in the cell regulation, proliferation . Quantithttp://mupro.proteomics.ics.uci.edu/) predicts that the mutations decrease protein stability.The c. 3125 A>G substitution occurs within the extracellular FNIII 11 domain that contains a recognition sequence in a flexible loop between two strands. These extracellular domains are able to bind ligands including extracellular proteins and other molecules such as collagen and heparin, respectively, as well as ligands on the cell –25. The PTPRQ are the pathogenic cause of ARNSHL in a non-consanguineous Chinese family. Identification of these two mutations in PTPRQ further confirms its crucial role in auditory function.In summary, our findings confirm that novel compound heterozygous mutations in http://www.textcheck.com/certificate/14r3OHThe English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis.C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions.The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.Our findings indicate that The online version of this article (doi:10.1186/s12864-016-2632-3) contains supplementary material, which is available to authorized users. Cyclospora cayetanensis is an emerging apicomplexan parasite related to Eimeria spp. [http://www.cdc.gov/parasites/cyclosporiasis/outbreaks/2015/index.html).ria spp. . After iria spp. , 2. Sincria spp.   and E. tenella (68.1 %). It has a gene density that is similar to that of E. tenella and T. gondii, but lower than that seen in some other apicomplexan parasites. In BLASTN analysis, we have identified the full mitochondrial and apicoplast genomes of C. cayetanensis [We obtained 120.9 million of 100-bp paired-end reads from Illumina sequencing and 960,078 reads of 400-450 bp from Roche GS-FLX 454 sequencing, yielding over 200-fold coverage of the genome. A total of 4811 contigs with an overall length of 46,816,962 bp were generated in the Eimeria spp. [C. cayetanensis genome , 1247 had one or more transmembrane regions, and 225 had a GPI-anchor attachment site. These numbers are similar to those in E. tenella and T. gondii , but several were from other organisms (n = 34)  of functional domains shared by apicomplexans and a smaller group (~546) by coccidia  Fig. . All ortdia Fig. . The hetlla Fig. .Fig. 2OrC. cayetanensis depends on carbon metabolism, including glycolysis, tricarboxylic acid (TCA) cycle and pentose phosphate pathways, for energy generation  rather than ADP-Glc used in green algae and land plants [E. tenella and C. cayetanensis have the unique ability to reversely produce mannitol from fructose. A similar pathway may be present in Cryptosporidium spp., although it utilizes mannose rather than fructose [P. falciparum and coccidia. Cryptosporidium spp. are able to convert UDP-Glc into UDP-GlcA (glucuronate) and then into UDP-Xyl (xylose).Similar to most other apicomplexans, d plants . All cocfructose . The amifructose . All apiC. cayetanensis and E. tenella possess neither the phosphoenolpyruvate (PEP) carboxylase utilized by P. falciparum and Cryptosporidium spp. nor the pyruvate carboxylase present in T. gondii [C. cayetanensis, E. tenella and other apicomplexans except Cryptosporidium spp., allowing them to continuously produce oxaloacetate to supplement the TCA cycle. In P. falciparum and T. gondii, the glycolysis and TCA cycle are disconnected due to the fact that the pyruvate dehydrogenase complex is localized in apicoplasts rather than mitochondria [T. gondii, suggesting that a partial TCA cycle exists in its apicoplast [C. cayetanensis and E. tenella. Thus, C. cayetanensis probably also possesses a partial TCA pathway in its apicoplast.Within the pyruvate metabolism, . gondii . Howeverchondria . In addiicoplast , 15. TheC. cayetanensis probably uses the pentose phosphate pathway to produce de novo phosphoribosyl pyrophosphate (PRPP), which is involved in pyrimidine biosynthesis. A ribokinase is present only in T. gondii, E. tenella and C. cayetanensis, suggesting that only coccidia are able to salvage ribose from the host in addition to synthesizing it de novo. Compared to P. falciparum and T. gondii, the deoxyribose phosphate aldolase for deoxyribose catalysis is absent in both E. tenella and C. cayetanensis. Another important intermediate within the pentose phosphate pathway, erythrose-4-phosphate, is the substrate in biosynthesis of shikimate as well as folate, which is eventually converted into tetrahydrofolate (THF) and methylene-THF. These two folates are essential for nucleotide conversion and amino acid conversion, respectively. In addition to the de novo synthesis of folates, apicomplexan parasites can transport folic acid from the extracellular environment using specific cytosol membrane transporter proteins (Table T. gondii possesses two extended sub-pathways for folate metabolism: 1) the biosynthesis of dihydrobiopterin and tetrahydrobiopterin, which can provide hydroxyl for converting phenylalanine to tyrosine; and 2) the biosynthesis of molybdopterin, the cofactor for sulfite oxidation [C. cayetanensis.Like most other apicomplexan parasites, ns Table . Furtherxidation . None ofC. cayetanensis genome, similar to E. tenella and T. gondii . However, all coccidia can probably synthesize putrescine reversely from spermine salvaged from the host.Among apicomplexans, only thionine . Due to de novo; they have to salvage them from the host through an amino acid transporter embedded in the plasma membrane [C. cayetanensis and E. tenella, however, each has only one amino acid transporter, compared with 10 in Cryptosporidium spp. and 6 in T. gondii -methyl-malonyl-CoA, respectively, supplementing intermediates for the TCA cycle [T. gondii and C. cayetanensis have the full set of enzymes for the degradation of propionyl-CoA, generating pyruvate and succinate. In addition, T. gondii has a pyruvate carboxylase, catalyzing pyruvate to oxaloacetate to make the methyl-citrate cycle a full pathway, similar to bacteria and fungi [No apicomplexans are able to synthesize aromatic amino acids membrane . C. cayeii Table . Some ofd L-DOPA . The genCA cycle . T. gondnd fungi .de novo and have to salvage them from the host  synthase in C. cayetanensis and E. tenella suggests that these parasites may have lost the ability to synthesize PLP, a component of vitamin B6, de novo from glutamine. However, the salvage pathways through the phosphorylation of pyridoxal or oxidation of pyridoxine/pyridoxamine phosphate are present in C. cayetanensis [de novo in the apicoplast, or salvaged from the host and utilized in the mitochondrion in T. gondii [C. cayetanensis. E. tenella lacks the enzymes used in mitochondria, but possesses dehydrogenase complexes similar to T. gondii and C. cayetanensis, suggesting that this inferred gene loss may not be true.Similar to tanensis . It has . gondii , 19. TheT. gondii and TA4-type surface antigens of E. tenella [C. cayetanensis and E. tenella  anchor for attachment to the plasma membrane, such as SRS (SAG1-related sequences) proteins of  tenella . Two esslla Fig. . In addiT. gondii and Cryptosporidium spp., C. cayetanensis does not add any glucose onto the core structure of the N-glycan precursor. In contrast, the enzymes that catalyze the addition of oligosaccharides onto the N-acetylglucosamines (GlcNAc) during N-glycan biosynthesis are absent in E. tenella, P. falciparum and B. bovis. During the trimming process, the glucosidase needed for removing the external glucose is absent in C. cayetanensis while another glucosidase involved in removing the remaining two glucoses is present.N-linked glycans, oligosaccharides attached to the asparagine (Asn) residue in a tripeptide sequence of Asn-X-Ser/Thr (where X is any amino acid except Pro) of proteins, are very common in eukaryotes . Based oN-acetylgalactosaminyltransferase , which transfers GalNAc from UDP-GalNAc to the hydroxyl group of specific serine or threonine residues in proteins [C. cayetanensis  adhesins involved in binding and interaction with host cells during the initial invasion, ii) secreted or membrane-associated peptidases involved in processing rhoptry and micronemal proteins of parasites and degrading proteins of the host, and iii) secreted signaling proteins such as protein phosphatases and kinases, which are injected across the plasma membranes into the host cell cytoplasm or nucleus, modulating host cell signaling pathways or immune responses to promote the survival of parasites . Some ofC. cayetanensis and E. tenella probably have an adhesive system very similar to that of T. gondii  are arrayed in four gene clusters. Their products, TA4-type surface antigens containing signal peptides and GPI-anchor sites, are thought to interact with host cell prior to invasion [C. cayetanensis genome. Only four putative TA4-type surface antigens, which are more similar to the subfamily SagA of Eimeria spp., were identified in C. cayetanensis and one of them has both a signal peptide and a GPI-anchor. The cysteine-rich secretory protein family (CAP), which TA4 surface antigens probably derived from, was also detected in the C. cayetanensis genome. By paralog analysis using OrthoMCL, a large group comprised of 31 genes annotated as hypothetical proteins were found in the C. cayetanensis genome. Some (n = 11/31) have cytosol membrane-related or periplasmic substrate binding-related functions  and some other proteins [C. cayetanensis and E. tenella suggesting that the motor structure may be conserved within all apicomplexans , is required for the processing of microneme proteins, affecting the efficiency of adhesion of tachyzoites [C. cayetanensis and E. tenella, the ortholog of another rhoptry subtilisin-like protease with specificity similar to the ROP1 mutarase [T. gondii and are known to play essential roles in the growth and intracellular survival of parasites [E. tenella, C. cayetanensis has four members of these two types of proteases. Even though the substrate for metalloproteinases, named toxolysins, is unclear, the presence of a rhoptry pro-domain cleavage site within toxolysin-1 (TLN1) suggests that toxolysins are probably protein maturases [T. gondii [C. cayetanensis, whereas homologs of TgROM2 and TgROM6 were not identified in E. tenella  secreted by the rhoptry and delivered into host cell nuclei during invasion [C. cayetanensis and E. tenella  to modulate host cell functions. The best known is TgROP18, which phosphorylates and inactivates host immunity-related GTPases [C. cayetanensis and E. tenella. E. tenella has a smaller number of ROPKs and several E. tenella-specific groups of ROPKs [C. cayetanensis genome, significantly smaller than the number in the E. tenella genome but similar to that in E. falciformis [C. cayetanensis are ROP21/27/35-like and E. tenella-specific ROPKs , with some similarities to the plant AP2 , 43. In C. cayetanensis shares some of the genomic features and metabolic capabilities of coccidia such as T. gondii and E. tenella. Compared with the metabolism in T. gondii, C. cayetanensis and E. tenella primarily lack de novo biosynthesis of certain amino acids and the ability to salvage amino acids directly from the host is significantly reduced. Differences in the degradation and hydroxylation pathways of some amino acids were also observed among the coccidian parasites examined. It appears amino acid metabolism evolves more rapidly in coccidia than other metabolic pathways. It is possible the lack of these amino acid metabolic pathways has reduced the target tissue range in C. cayetanensis and E. tenella. The only unique metabolic pathway present in C. cayetanensis and E. tenella but absent in T. gondii is the synthesis of mannitol from fructose catalyzed by a single enzyme. The fungi-like mannitol cycle metabolism (fructose-mannitol-mannitol phosphate-fructose phosphate-fructose) was known to be present in E. tenella [C. cayetanensis oocysts. A clear difference between C. cayetanensis and E. tenella is the ability of the former to degrade propionyl-CoA to produce pyruvate and succinate, probably as supplements for the TCA cycle, suggesting that C. cayetanensis probably has more carbon sources for mitochondrial metabolic activities than E. tenella.Comparative genomic analysis indicates that  tenella . Mannito tenella . It is pC. cayetanensis and Eimeria spp. Because of the secondary loss of ALG enzymes, the length of N-glycan precursors is divergent among apicomplexans, from 10 sugars in T. gondii to 2 in E. tenella and none in Theileria spp. [C. cayetanensis possesses 7 sugars, Man5GlcNAc2. Similarly, a paucity of enzymes involved in the biosynthesis of GPI-anchor was detected in C. cayetanensis and E. tenella. Both C. cayetanensis and E. tenella lack mannosyltransferases PIG-V and PIG-B. In addition, phosphomannomutase and dolichol-phosphate-mannose (Dol-P-Man) synthase, the enzymes involved in generation of Dol-P-Man, which is the substrate for N-glycan and GPI-anchor synthesis, are absent in Eimeria spp. [C. cayetanensis. Thus, there are substantial differences in both GPI anchor biosynthesis and N-glycosylation between C. cayetanensis and Eimeria spp.The mechanisms involved in post-translational modifications of proteins may be different between ria spp. . The N-gria spp.  but are C. cayetanensis has host cell receptors and invasion process similar to that of T. gondii and E. tenella: gliding powered by an actin motor before invasion, interactions with the host cell through surface antigens, forming a moving junction with a series of secreted proteins to initiate invasion, forming a PV structure inside the host cell, and secreting various protein kinases, protein phosphatases, and other catalytic proteins to modify the host metabolic pathways for the evasion of host immune responses. The amplification and diversification of surface and secreted proteins are probable determinants for distinct transmission, host range and pathogenicity of various coccidia [T. gondii, the dramatic reduction in protein kinases and phosphatases suggests that C. cayetanensis and E. tenella have only limited capacity to regulate host cell signaling pathways and gene expression, or use a divergent system to do so. These genomic characteristics of E. tenella and C. cayetanensis are probably responsible for their exclusive enteric life cycle. Between C. cayetanensis and E. tenella, the former has a further reduction in the number and type of ROPKs.The similar repertoire of host cell invasion-related proteins possessed by all coccidian parasites suggests that coccidia . CompareT. gondii invasion [N. caninum possesses the same type of SRS proteins seen in T. gondii, but has a significant increase in their number [C. cayetanensis nor E. tenella has this family of surface proteins. In contrast, Eimeria spp. have the unique TA4-type surface antigens and show divergence in their compositions among species [C. cayetanensis genome, we detected several TA4-type surface antigen coding genes in different genomic regions. Thus, surface antigens are probably the most rapidly evolved proteins in coccidia and are likely determinants for host specificity. We assume that C. cayetanensis possesses its own unique surface antigens. The paralogous genes we identified in this study encode mostly hypothetical proteins, one of which has sequence homology to the PfEMP1 of P. falciparum. They probably represent the surface antigens of C. cayetanensis, as many of these proteins are predicted to have membrane-related or periplasmic substrate binding-related functions. Further studies on the expression, localization and neutralization ability of these proteins are needed to confirm their surface antigen nature in C. cayetanensis.Surface antigens SRS (SAG1-related sequences) are involved in initial interaction with host cells in invasion . N. canir number . Neither species . In the C. cayetanensis probably possesses a classical coccidian metabolism and has a host cell invasion system very similar to Eimeria spp. and T. gondii. The amino acid metabolism and post-translation modifications of proteins are probably the most rapidly evolved metabolic pathways among coccidia. Compared with the heteroxenous T. gondii, the monoxenous C. cayetanensis and Eimeria spp. appear to have very limited abilities or use different mechanisms to modulate host nuclear activities and signaling pathways during invasion. The dominant surface antigens seen in other coccidia are not present or are significantly reduced in number in C. cayetanensis and the presence of divergent surface proteins among coccidia suggests that these proteins are likely determinants of host specificity. These observations, however, are based on results of comparative genomic analyses and need to be validated by functional studies. Overall, the availability of whole genome sequence data has significantly improved our understanding of the biology of C. cayetanensis and may facilitate the development of molecular diagnostic tools for traceback studies of foodborne cyclosporiasis outbreaks.Through whole genome sequencing and comparative genomic analysis, we have shown that C. cayetanensis specimen sequenced in this study was collected in July 2011 from a patient with severe diarrhea in Kaifeng, Henan, China, where cyclosporiasis is endemic and C. cayetanensis isolates were characterized morphologically and by sequence analysis of the SSU rRNA gene [C. cayetanensis by ultraviolet epifluorescence microscopy and PCR analysis of a ~680-bp fragment of the SSU rRNA gene [C. cayetanensis oocysts were purified from the specimen using sucrose and cesium chloride gradients [6 oocysts using a QIAamp®DNA Mini Kit , after the oocysts were subjected to five freeze-thaw cycles and overnight digestion with proteinase K. About 100 ng of extracted DNA was amplified using REPLI-g Midi Kit  according to the manufacturer-recommended procedure.The RNA gene . It was RNA gene . DNA seqradients  and furtC. cayetanensis isolate was sequenced on a Roche 454 GS-FLX Titanium System  using the standard Roche library protocol, and on an Illumina Genome Analyzer IIx and a Hiseq 2500  using the Illumina TruSeq (v3) library protocol. For Roche 454 sequencing, sequence reads of approximately 400 bp were generated in one run and 450 bp in another, whereas in Illumina sequencing, 100 × 100 bp paired-end reads were generated. The raw sequencing reads from the two platforms were combined, and reads of quality score below 30 were trimmed using CLC Genomics Workbench 7.03 (http://www.clcbio.com/products/clc-genomics-workbench). They were assembled into contigs using the default parameters.The T. gondii, E. tenella and C. cayetanensis and assess the completeness of the genome sequencing. Simple tandem repeat and low complexity sequences in the C. cayetanensis genome were identified using RepeatMasker version 4.0.3 (http://repeatmasker.org/), whereas LTR-retrotransposons were identified using LTRharvest [The BLASTN  program Rharvest . Circos Rharvest  was usedhttp://hmmer.janelia.org/) was used to search chromodomain (PF00385) and reverse transcriptase (PF00078) motifs in these sequences using the HMM model from Pfam [http://pfam.xfam.org/). A cluster analysis of all LTR-retrotransposons was conducted based on nucleotide sequence identities. The longest LTR-retrotransposon from the biggest group was used for phylogenetic analysis. The chromovirus-type LTR sequence of E. tenella was randomly chosen and other LTR-retrotransposons were retrieved from NCBI GenBank. ClustalX v2 [All predicted LTR-retrotransposons were extracted and translated into amino acid sequences. HMMER , while GeneMark-ES is a self-training gene predictor. After examination of outcomes of gene predictions (data not shown), we kept all genes predicted by AUGUSTUS, because they fit well into the gene model. New genes predicted by both SNAP and GeneMark-ES were combined with the results of AUGUSTUS as the final protein-coding gene set of C. cayetanensis. SignalP v4.1 [Protein-encoding genes in the TUS v2.7 , SNAP [6TUS v2.7 , and GenTUS v2.7 . AUGUSTUalP v4.1  and TMHMalP v4.1  with defalP v4.1 . GPI ancalP v4.1 .http://eupathdb.org/eupathdb/).A BLASTP  search oC. cayetanensis were identified by using OrthoMCL [http://bioinfogp.cnb.csic.es/tools/venny/index.html). The phylogenetic relationship between C. cayetanensis and common apicomplexans was assessed by neighbor-joining analysis of an alignment of concatenated protein sequences of 100 orthologs, as described by Woo [Orthologs of other apicomplexans in the predicted proteome of OrthoMCL . Groups OrthoMCL . Venn did by Woo . Gblocksd by Woo  was usedT. gondii, N. caninum, E. tenella, and other apicomplexans into 42 distinct subfamilies, was used in the prediction and analysis of ROPKs in C. cayetanensis with the best hit score threshold set at 100. All putative ROPKs sequences identified in E. tenella, E. falciformis, and C. cayetanensis were extracted and analyzed with the neighbor-joining method described above.A comparison of transporter proteins was conducted based on the Pfam search results. The database for coccidia-specific rhoptry kinases and pseudokinases HMM profiles , which cThe genome sequencing was done on a delinked residual diagnostic specimen. The work was covered by Human Subjects Protocol No. 990115 “Use of residual human specimens for the determination of frequency of genotypes or sub-types of pathogenic parasites,” which was reviewed and approved by the Institutional Review Board of the Centers for Disease Control and Prevention (CDC). No personal identifier was associated with the specimen at the time of its submission for diagnostic service at CDC.http://purl.org/phylo/treebase/phylows/study/TB2:S19120).The datasets supporting the conclusion of this article, including all Sequence Read Archive (SRA) data (SRX665300 and SRX681889), assembled contigs (ASM76915v1), and annotations (JROU00000000) are available in the NCBI BioProject under the accession No. PRJNA256967. The phylogenetic data supporting the conclusions of this article are available in the TreeBase (