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Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria, occurring in many, but not all, genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, and Yersinia enterocolitica. T2S-expressing plant pathogens include Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa. T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection.
Secreted proteins have a major role in the pathogenesis of bacterial infections, including important diseases of humans, animals, and plants. In the case of Gram-negative bacteria, there are seven secretion systems (types I, II, III, IV, V, VI, and IX) that mediate the export of “effector” proteins out of the bacterial cell and into the extracellular milieu or into target host cells (1–3). Type II secretion (T2S) was the first such system to be defined, based upon work done in the mid-1980s on pullulanase secretion by Klebsiella oxytoca (4). Further insight into T2S was then gained from the examination of Aeromonas, Pseudomonas, Vibrio, and a few additional members of the gammaproteobacteria (5, 6). Thus, T2S is considered a two-step process; i.e., proteins to be secreted are first carried across the inner membrane (IM) and into the periplasm by the Sec translocon (7) or Tat pathway (8) and then, after folding into a tertiary conformation (and in some instances, undergoing oligomerization), are transported across the outer membrane (OM) by the dedicated T2S apparatus (2). The T2S machinery is made up of 12 “core” proteins, which are denoted here as T2S C, D, E, F, G, H, I, J, K, L, M, and O (9, 10). In recent years, there has been remarkable progress toward elucidating the precise structure of the T2S apparatus (2, 11–16). In essence, there are four subcomplexes: (i) an OM “secretin,” which is a pentadecamer of the T2S D protein that provides a pore through the membrane; (ii) an IM platform composed of T2S C, F, L, and M, with T2S C providing a connection to the OM secretin; (iii) a cytoplasmic ATPase, which is a hexamer of T2S E that is recruited to the IM platform; and (iv) a periplasm-spanning pseudopilus which is a helical filament of the major pseudopilin T2S G capped by the minor pseudopilins T2S H, I, J, and K. Finally, T2S O is an IM peptidase that cleaves and methylates the pseudopilins as a prelude to their incorporation into the pseudopilus. Thus, during T2S, protein substrates present in the periplasm are delivered to the T2S apparatus, presumably following their recognition by T2S C and T2S D (17), and then using energy generated at the IM, the pseudopilus acts as a piston or an Archimedes screw to push the proteins through the OM secretin (2). Although recent papers have detailed the structure of the T2S apparatus and the molecular mechanism of secretion (11–15), it has been some time since there was a review focused on the prevalence of T2S and its role in pathogenesis. Hence, this minireview will provide an update on the types of bacteria and pathogens that have T2S, the numbers and kinds of proteins that are secreted via T2S, and how T2S-dependent proteins promote infection.
Prevalence of type II protein secretion systems.Following the advent of whole-genome sequencing, complete or nearly complete sets of T2S genes (i.e., containing all or almost all of the core constituents, T2S CDEFGHIJKLMO) were identified in 32 genera of Proteobacteria, comprising 22 genera in the gammaproteobacteria, 4 genera each in the alpha- and betaproteobacteria, and 2 genera in the deltaproteobacteria (10, 18). However, T2S genes were absent from 29 other genera of Proteobacteria, including those in the epsilonproteobacteria, indicating that T2S occurs in many, but not all, genera in the phylum Proteobacteria (10). Extending this analysis, a recent study, which defined the full set of T2S genes as one encoding T2S CDEFGHIJKLMNO, identified the system in 360 of the 1,528 Gram-negative genomes examined, with 58%, 45%, 15%, 6%, and 0% prevalence among beta-, gamma-, delta-, alpha-, and epsilonproteobacteria, respectively (19). Figure 1 depicts the distribution of T2S within the evolutionary tree of the Proteobacteria. Looking beyond the Proteobacteria, there are interesting examples of organisms that have a smaller number of T2S-related genes; e.g., Leptospira interrogans of the Spirochaetes encodes T2S CDEFGJKLMO, Chlamydia and Chlamydophila species within the Chlamydiae harbor genes for T2S CDEFG, Rhodopirellula baltica belonging to the Planctomycetes may encode T2S DEFGIKO, Aquifex aeolicus of the Aquificae carries homologs for T2S DEFGO, and Thermotoga maritima of the Thermotogae appears to encode T2S DEFG (10, 19–22). In the case of Chlamydia trachomatis, one of these genes has been linked to protein secretion (20), suggesting that there may be different subclasses of T2S that deviate from the canonical system present in the Proteobacteria. In Synechococcus elongatus belonging to the Cyanobacteria, a T2S E-like gene has been linked to protein secretion; however, this gene may be encoding a component of a type IV pilus rather than a T2S apparatus (19, 23). So far, genome database analyses have failed to reveal any evidence for potential T2S systems in Bacteroidetes, Chlorobi, Fusobacteria, or Verrucomicrobia (10, 19). Thus, despite the fact that T2S is often referred to as the main terminal branch of the general secretory pathway (5, 24, 25), T2S is not, by any means, conserved among Gram-negative (“diderm”) bacteria. Rather, in its canonical form, T2S is largely restricted to the Proteobacteria (Fig. 1). Furthermore, even in the Proteobacteria, T2S, though common, is not universal. Put another way, T2S may be no more prevalent across Gram-negative genera than is type I, III, IV, V, or VI secretion (19). Hence, T2S is best considered a specialized secretion system that a subset of Gram-negative bacteria has evolved to utilize for their growth within the environment or larger hosts. Table 1 shows a comprehensive list of those bacteria in which T2S has been shown by mutational analysis to actually be functional.
Representative distribution of T2S genes among the Proteobacteria. An unrooted phylogenetic tree of the Proteobacteria and several other bacteria was constructed with aligned 16S rRNA sequences (65, 66) using standard neighbor-joining methods (67, 68). Genus names are denoted at each leaf. Clades representing the alpha-, beta-, gamma-, delta-, and epsilonproteobacteria are identified by the α, β, γ, δ, and ε Greek symbols. The bar represents the number of nucleotide substitutions per site. Bacteria that have been demonstrated to express a functional T2S system are indicated in green. Representative bacteria that have a complete or nearly complete set of T2S genes but for which functionality has not yet been shown are indicated in red. Representative bacteria that lack T2S genes are indicated in black.
T2S in pathogens of humans and animals.The human pathogens that are known to possess functional T2S include representatives from 10 genera of gammaproteobacteria (Acinetobacter, Aeromonas, Escherichia, Klebsiella, Legionella, Photobacterium, Pseudomonas, Stenotrophomonas, Vibrio, and Yersinia), one genus of betaproteobacteria (Burkholderia), and one genus of Chlamydiae (Chlamydia) (Fig. 1). Among the prominent human pathogens that use T2S are Acinetobacter baumannii, Aeromonas hydrophila, Burkholderia cenocepacia, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica (Table 1). Some of these T2S-expressing bacteria are also natural pathogens of animals, ranging from those afflicting fish (e.g., A. hydrophila, Aeromonas salmonicida, Photobacterium damselae, and V. anguillarum) to those impacting other mammals (e.g., B. pseudomallei and Y. enterocolitica). Based upon genome sequencing and Southern blot analyses, it is likely that additional pathogenic members of these genera employ T2S (10, 26–28). In most cases, these human and animal pathogens encode a single T2S system. Yet, for some strains of E. coli, P. aeruginosa, S. maltophilia, and Y. enterocolitica, there are two or three distinct T2S systems (Table 1). As more isolates are sequenced, there will likely be additional examples of multiple sets of T2S genes. At present, the functionality of the second system in E. coli, S. maltophilia, and Y. enterocolitica is unknown, as no secreted substrates or activities have been defined. A specialized growth condition(s) may be needed in order for the expression of a T2S system to be evident; e.g., whereas expression of the Xcp T2S system of P. aeruginosa is easily observed in bacteriological media, expression of the Hxc system occurs only in low phosphate. Currently, there is quite a range in the size of the T2S output of the T2S-expressing pathogens, going from one protein or activity as in K. pneumoniae, Pseudomonas alcaligenes, and V. parahaemolyticus to dozens as in Acinetobacter nosocomialis, B. pseudomallei, L. pneumophila, P. aeruginosa, and V. cholerae (Table 1). The output of many, if not all, T2S systems, however, will likely prove to be greater once proteomic analysis is applied. Most studies have identified T2S-dependent proteins in culture supernatants; however, there is increasing evidence that some substrates remain bound to the bacterial surface after secretion. The first such example was the pullulanase of K. oxytoca, and further examples have now been found in E. coli, P. aeruginosa, and V. vulnificus (Table 1). The mechanism by which the T2S apparatus facilitates the anchoring of proteins to the bacterial outer surface rely on acylation and hydrophobic or polar interactions (29). Nonetheless, by virtue of their surface localization, these proteins can be present on outer membrane vesicles (OMVs) that bleb from the bacterial cell surface (30). Other T2S substrates come to reside within OMVs, as a result of their localization in the periplasm prior to transport across the OM by the T2S apparatus (30). Because of their fusogenic capability, OMVs provide an alternative means for delivering T2S-associated substrates to host targets.
Collectively, the human pathogens that express T2S are responsible for a wide variety of diseases, ranging from pneumonia (A. baumannii, L. pneumophila, K. pneumoniae, P. aeruginosa, and S. maltophilia) to gastroenteritis and diarrhea (E. coli, V. cholerae, and Y. enterocolitica) to bloodstream (A. hydrophila, B. pseudomallei, and V. vulnificus), urinary tract (E. coli), and genital tract (C. trachomatis) infections (Table 1). Furthermore, these bacteria include both extracellular (Acinetobacter, Aeromonas, Escherichia, Klebsiella, Pseudomonas, Stenotrophomonas, and Vibrio species) and intracellular (Burkholderia species, C. trachomatis, L. pneumophila, and Y. enterocolitica) pathogens. These facts imply that T2S facilitates disease in a variety of ways and is not limited to a particular pathogenic event or site of infection. Support for this inference derives from the many types of degradative enzymes and toxins that are secreted by T2S; i.e., ADP-ribosylating enzymes, carbohydrate-degrading enzymes, lipolytic enzymes, nucleases, pore-forming proteins, phosphatases, peptidases, and proteases (Table 1). Particularly well-known examples of T2S-dependent substrates are cholera toxin produced by V. cholerae, exotoxin A of P. aeruginosa, and heat-labile (LT) toxin from enterotoxigenic E. coli. The most direct proof for the role of T2S in pathogenesis is based upon the attenuated virulence of T2S mutants in animal models of disease, as has been shown for A. baumannii, A. hydrophila, B. cenocepacia, B. pseudomallei, E. coli, K. pneumoniae, L. pneumophila, P. aeruginosa, V. vulnificus, and Y. enterocolitica (Table 1). Additional assays using these mutants and/or isolated secreted proteins have revealed a diversity of mechanisms by which T2S facilitates disease. These mechanisms include the death of host cells by lysis or toxicity, degradation of tissue and extracellular matrix, cleavage of defense molecules such as cytokines and complement components and other means of suppressing innate immunity, adherence to epithelial cell surfaces, disruption of the tight junctions between host cells, biofilm formation, invasion into host cells or subsequent intracellular growth, deubiquitination, iron acquisition, and other forms of nutrient assimilation, and alterations in host ion flux triggering diarrhea (Table 1). Undoubtedly, there are even more ways in which T2S promotes pathogenesis; e.g., proteomic analysis has revealed a number of T2S substrates that are “novel,” having no sequence similarity to known proteins or enzymes (e.g., L. pneumophila, P. aeruginosa, and V. cholerae) (Table 1).
T2S in pathogens of plants.T2S systems are also present and functional in plant pathogens that belong to the gammaproteobacteria (Dickeya, Erwinia, Pectobacterium, Xanthomonas, and Xylella) and betaproteobacteria (Burkholderia and Ralstonia) (Fig. 1). The T2S-expressing phytopathogens include Burkholderia gladioli, Burkholderia glumae, Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Pectobacterium wasabiae, Ralstonia solanacearum, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa (Table 1). Collectively, they cause serious diseases of flowers, fruit (e.g., pear, citrus, and grape), rice, tubers, and vegetables (e.g., crucifers and peppers) (Table 1). Many of the concepts noted above when discussing the T2S-expressing human pathogens also apply here. For example, some of the plant pathogens have multiple T2S systems (D. dadantii, X. campestris pv. vesicatoria), secrete ≥15 T2S substrates (B. glumae, D. dadantii, P. carotovorum, and R. solanacearum), and express T2S substrates on their surface (D. dadantii). They also secrete some enzymes that are similar to those made by the human and animal pathogens (e.g., lipases and proteases) as well as “novel” proteins that may encode a new enzymatic activity and/or mediate a new type of process. Not surprisingly, the T2S systems of the phytopathogens elaborate a large number and variety of carbohydrate-degrading enzymes that specifically degrade plant tissue, e.g., cellulases, pectate lyases, xylanases, and polygalacturonases (Table 1). In every case examined, mutations in the genes encoding T2S diminish virulence in a relevant host(s) (Table 1), clearly showing the importance of T2S in plant pathogenesis.
T2S in nonpathogenic, environmental bacteria.Although T2S in pathogens has received the greatest attention, there have been a number of studies documenting T2S functionality in nonpathogenic bacteria or bacteria that only very rarely cause disease (Table 1). These bacteria are quite diverse, ranging from alphaproteobacteria (Caulobacter and Gluconacetobacter) to betaproteobacteria (Cupriavidus and Ralstonia) to gammaproteobacteria (Aeromonas, Cellvibrio, Escherichia, Marinobacter, Methylococcus, Pseudoalteromonas, Pseudomonas, Shewanella, and Vibrio) to deltaproteobacteria (Geobacter) (Fig. 1). In most cases, they are primarily free-living organisms, inhabiting soil, freshwater, and/or salt water. However, some exist in symbiotic relationships with plants (Gluconacetobacter diazotrophicus and Pseudomonas fluorescens) or animals (Aeromonas veronii, E. coli, and Vibrio fischeri), and in the case of A. veronii, T2S actually promotes the symbiosis with leeches (Table 1). Based on the genome database, it is likely that many more nonpathogens utilize T2S, including species of Azoarcus, Bdellovibrio, Bradyrhizobium, Chromobacterium, Mesorhizobium, Methylotenera, and Myxococcus as well as marine gammaproteobacteria belonging to Idiomarina, Marinomonas, Psychromonas, and Saccharophagus (10, 18, 31–33) (Fig. 1). The study of nonpathogens has revealed a variety of secreted proteins and processes that had not been seen with the pathogenic organisms. Among the novel T2S-dependent substrates are the multicopper oxidase of Geobacter sulfurreducens, levansucrase of G. diazotrophicus, c-type cytochrome of Methylococcus capsulatus, Mn-oxidizing enzymes of Pseudomonas putida, and NAD-glycohydrolases of V. fischeri, and included in the T2S-facilitated processes are pigmentation by Pseudomonas tunicata and Fe3+ reduction and extracellular respiration by Shewanella oneidensis (Table 1). Thus, by considering the full range of T2S-expressing bacteria, the functional diversity of T2S can be even better appreciated.
T2S in the transition of environmental bacteria to pathogens, as illustrated by V. cholerae and L. pneumophila.Nearly all of the T2S-expressing pathogens exist in the environment in addition to their higher organism hosts. Arguably, the impact of T2S is best appreciated by contemplating how T2S assists bacteria in both their environmental niche(s) and their human, animal, or plant host(s). This point is most clear from studies done with V. cholerae, the agent of cholera and a classic extracellular pathogen, and L. pneumophila, the etiologic agent of Legionnaires' disease and a well-known intracellular pathogen. In the case of V. cholerae, the Eps T2S system enhances attachment to and biofilm formation on abiotic and biotic surfaces in marine environments (34). This, in turn, promotes the growth of planktonic V. cholerae as well as bacterial colonization of marine creatures such as bivalves, copepods, and cladocerans (35). Among the T2S-dependent proteins that mediate environmental persistence are the chitin-binding protein GbpA that aids in attachment, ChiA and other chitinases that generate carbon and energy sources for growth, the biofilm-promoting RbmC, and the HapA protease which can degrade the matrix that covers the eggs of chironomids (34–37). By helping to increase the numbers of V. cholerae in the environment, T2S promotes the transmission of the Vibrio pathogen to human hosts via the ingestion of contaminated waters. Once in the human host, T2S continues to play a major role by secreting HapA which degrades mucin and thereby permits bacterial access to the underlying intestinal epithelium, GbpA which enhances binding to mucins that overlay the epithelium, cholera toxin which triggers water efflux from enterocytes (i.e., massive watery diarrhea), and HapA, VesA, and VesB which can proteolytically activate cholera toxin and other toxins (34, 35, 38–41). In summary, T2S is unquestionably important for V. cholerae both in its natural marine environment and in the human host, facilitating, in multiple ways, extracellular replication and dissemination (Fig. 2A).
Roles of T2S in V. cholerae and L. pneumophila. (A) More than 20 proteins are secreted via the T2S system of V. cholerae. T2S promotes the environmental survival of extracellular V. cholerae in a variety of ways, including the colonization of biotic surfaces (left side, in blue). This facilitates transmission to the human host, where T2S mediates another set of activities that leads to cholera (right side, in red). (B) More than 25 substrates are handled by the T2S system of L. pneumophila. In the environment, T2S facilitates the spread of L. pneumophila by contributing to planktonic survival, biofilm formation, and intracellular infection of amoebae (left side, in green). Following the inhalation of L. pneumophila, T2S promotes bacterial growth within lung macrophages, which leads to tissue damage and pneumonia (right side, in red).
Turning to L. pneumophila, it is necessary to first emphasize that the persistence of the Legionella pathogen in freshwater environments is primarily due to its capacity to infect a wide array of amoebae (42). The Lsp T2S system of L. pneumophila has a major role in infection of amoebae, promoting intracellular growth in at least four genera, i.e., Acanthamoeba, Naegleria, Vermamoeba (formerly Hartmannella), and Willaertia (43–47). This function of T2S is manifest over a temperature range of 22 to 37°C, further indicating the impact of T2S across different aquatic niches (48). The T2S-dependent substrates that are known to potentiate amoebal infection are the acyltransferase PlaC, metalloprotease ProA, RNase SrnA, and novel proteins NttA and NttC (46, 47, 49, 50). Interestingly, the importance of each of these secreted proteins varies depending upon the amoeba being infected, suggesting that the T2S repertoire of L. pneumophila has evolved, in part, to enhance the bacterium's broad host range (47). Besides its predilection for amoebae, L. pneumophila survives extracellularly in its aquatic habitats, either planktonically or in multiorganismal biofilms (51, 52). T2S is also relevant for these lifestyles, as documented in several ways. First, T2S mutants display impaired extracellular survival in tap water samples when incubated at 4 to 25°C (48). The fact that the secretome of L. pneumophila changes with temperature suggests that one or more secreted proteins, including a predicted peptidyl-prolyl cis-trans isomerase (PPIase), facilitate low-temperature survival (53). Second, a mutant specifically lacking the T2S-dependent Lcl protein exhibits a reduced ability to form biofilms (54). Finally, T2S mutants demonstrate impaired sliding motility, which is linked to the secretion of a novel surfactant (55–57). By fostering L. pneumophila growth within water systems, T2S contributes to the genesis of human infection which occurs via the inhalation of contaminated water droplets generated by various aerosol-generating devices. Yet, T2S also enhances L. pneumophila growth within the lung itself; i.e., secretion mutants are impaired in both murine and guinea pig models of pneumonia (26, 43, 58). The intrapulmonary role of T2S primarily involves L. pneumophila intracellular infection of macrophages (26, 59). Recent studies indicate that T2S is not required for L. pneumophila entry into the macrophage host or its subsequent evasion of phagosome-lysosome fusion (60). Rather, T2S facilitates the onset of bacterial replication at 4 to 8 h postentry as well as the capacity to grow to large numbers within the Legionella-containing vacuole at 12 h and beyond. This growth promotion involves both the retention of the host GTPase Rab1B on the Legionella-containing vacuole as well as a Rab1B-independent event(s) that is yet to be defined (60). Besides facilitating bacterial growth in macrophages, T2S is necessary for optimal replication within epithelial cells, which likely are a secondary host cell during lung infection (59). Furthermore, the T2S system dampens the cytokine output of infected macrophages and epithelial cells (59). This suppression of the innate immune response, which is manifest at the transcriptional level due to dampening of the MyD88 and Toll-like receptor 2 signaling pathway, is believed to initially limit inflammatory cell infiltrates into the lung and thus permit prolonged bacterial growth (61). As for the T2S-dependent proteins that are known to potentiate disease, the chitinase ChiA promotes bacterial growth and persistence in the lungs but in a manner that appears to be independent of intracellular growth (58). One hypothesis for this novel finding is that ChiA acts upon chitin-like molecules (e.g., O-GlcNAcylated proteins) in the lung. Finally, the metalloprotease ProA functions as a virulence factor by degrading lung tissue and cytokines (59, 62–64). Thus, L. pneumophila provides a striking example of the many ways in which T2S can promote both bacterial growth in the environment and virulence in the human host (Fig. 2B). L. pneumophila's adaptation to an intracellular niche in aquatic amoebae engendered it with the capacity to grow in human macrophages, and it is now clear that T2S plays a major role in both forms of intracellular infection.
Final thoughts and ongoing questions.In recent years, we have experienced an impressive increase in knowledge about bacterial T2S. These advancements include not only the fine-structure analysis of the T2S apparatus but also, as detailed here, a refined understanding of the distribution of T2S among Gram-negative organisms and the large and diverse roles of this secretion system (Table 1). Given the breadth of its involvement in pathogenic processes, it is clear that the importance of T2S rivals that of the other known secretion systems operating in Gram-negative bacteria that afflict humans, animals, or plants. Although we have learned a great deal about the output and functional consequences of T2S in pathogens and nonpathogens, there is still much insight to be gained, given that many of the secreted factors produced by these bacteria are still only minimally defined or entirely uncharacterized (Table 1). Indeed, some of these T2S-dependent substrates may represent new types of enzymes which might mediate novel pathogenic activities. Based on the data assembled in Table 1, there are also a number of T2S systems that are only slightly characterized and/or not yet examined in pertinent disease models. Moreover, the genome database indicates that there are many other bacteria, including pathogens, that harbor T2S systems that have not been investigated at all. All of these studies should take into consideration how the output and function of a T2S system might change depending upon growth conditions and regulatory networks. As the T2S catalog expands, various comparisons between the secreted proteins might reveal new structural similarities or motifs that help address a long-standing question in the field, that is, how T2S substrates are recognized by the secretion apparatus. In light of the now-demonstrated importance of T2S in a wide range of pathogenic bacteria, future work should also consider using the structural and functional knowledge gained to develop potential new strategies or reagents for preventing or combatting human, animal, or plant infections.
We thank past and present members of the Cianciotto lab for their studies on type II secretion and much helpful advice.
This work was funded by NIH grant AI043987 awarded to N.P.C.
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text:
Ключевые слова: quality of life, health, physical activity.
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text:
Present: DUFFLY, SMITH, & COHEN, JJ.
Firearms. Evidence, Firearm, Constructive possession. Constitutional Law, Assistance of counsel. Practice, Criminal, Assistance of counsel.
INDICTMENTS found and returned in the Superior Court Department on April 27, 2007.
The cases were tried before Frank M. Gaziano, J.
Ryan McLaughlin (Helle Sachse, Assistant District Attorney, with him) for the Commonwealth.
SMITH, J. The defendant, Hal McIntosh, appeals from his convictions on three indictments charging him with (1) possession of a firearm, second and subsequent offense; (2) possession of ammunition; and (3) possession of a loaded firearm. On appeal, the defendant claims that the judge committed reversible error in denying his motion for a required finding of not guilty on all three indictments. The defendant also contends that his trial counsel was constitutionally ineffective.
1. Evidence at the trial. Because the defendant challenges the sufficiency of the evidence, we summarize in detail the evidence, considering it in the light most favorable to the Commonwealth. Commonwealth v. Rodriguez, 456 Mass. 578 , 579 (2010). On January 7, 2007, Officer Sean Rooney was in a marked police cruiser and in uniform, patrolling an area in the Mattapan section of Boston. He was alone in his cruiser but was accompanied by another marked cruiser, operated by Officer Charlie Daniels. At about 6:30 P.M., the two police officers drove by 43 Harvard Street and observed two males standing outside on the top front step of the building. One was later identified as Daishaun Brown; the other was later identified as the defendant. The defendant made eye contact with Officer Rooney and then immediately turned and opened the outer door of 43 Harvard Street, but was unable to enter the building.
The officers parked their cruisers and spoke to the individuals. Officer Rooney spoke to the defendant, the other officer with Brown. Officer Rooney asked the defendant "if he lived there and needed help with regard to getting access." Both individuals seemed a little nervous and a little apprehensive but the defendant was not the subject of a patfrisk at that time. In answer to Officer Rooney's questions, the defendant stated that he did not live at that house, but was waiting for someone. At Officer Rooney's request, the defendant produced an identification card, handing it to the officer with his left hand. At that time, the defendant was also holding a cellular telephone, "scrolling through and using the walkie-talkie feature." While Officer Rooney was copying the defendant's identification card information, the defendant placed a full-fingered baseball batting glove on his right hand.
As Officer Rooney finished copying the defendant's information, the defendant went to the front door of the building and opened it. Officer Rooney told the defendant, "[D]on't go too far away. I still have your license. Come back and grab it. I'm all set with it." The defendant again reached out with his left hand and took back his identification card.
At that point, the owner of the building, Ms. Lewis, opened the door of the first-floor apartment that had been locked, came out, and asked, "What's going on?" Ms. Lewis was then asked, apparently by the defendant, "Is Seyon there?" Ms. Lewis told the defendant that she did not know him (the defendant) and that "there's no one here." The defendant responded, "Well, Seyon has my thing, and I need to get it." [Note 1] He then pushed Ms. Lewis to one side, entered the first-floor apartment, and went down a hallway, followed by Ms. Lewis and Officer Rooney. The defendant entered a bedroom, followed by Ms. Lewis. She saw the defendant bending by the middle part of a bed, but she could not see what he was doing. Ms. Lewis left the room to get Officer Rooney and the defendant closed the door. Ms. Lewis came back with the officer and attempted to open the door, but could not. Officer Rooney also tried, unsuccessfully at first, to get the door open. He could tell that the door was being held shut from the inside while he was trying to push it open. Officer Rooney finally pushed the door open and found the defendant standing directly behind it, within arm's reach of the door. Officer Rooney immediately pat frisked the defendant and, not finding any weapon on him, brought him outside to the front stairs and ordered him to sit there.
Officer Rooney went back to the bedroom where he was informed that Ms. Lewis's grandson, Kamau, occupied the room and was taking a shower. Officer Rooney suggested that they shut the door, wait for Kamau to return, and have him inspect his room to see if anything was missing, or if there was something in the room that should not be there.
years. The defendant was also a friend of Seyon Marshall, Kamau's cousin. See note 1, supra. Kamau stated that no one should be going into his room in his absence. At Officer Rooney's direction, Ms. Lewis, Kamau, and another police officer entered the room. Ms. Lewis asked Kamau to lift the mattress off the bed where the defendant was seen bending. A loaded handgun was found under the mattress. It was located near the foot of the mattress, an arm's length from the bedroom door. Kamau had last looked under the mattress the day before and had not seen a gun at that time. The firearm bore a single fingerprint of an unknown person.
The defendant did not testify at trial but through cross-examination and the testimony of the defendant's companion, Brown, offered the theory that he neither had the firearm on his person nor placed it under the mattress and that another individual, possibly Kamau, placed the gun there.
2. Sufficiency of the evidence. At trial, the Commonwealth proceeded on the theory that at the time the defendant was first confronted by Officer Rooney, the defendant was in actual possession of the firearm. Because the defendant was afraid that Office Rooney would discover the firearm, the defendant ran into the bedroom and placed the firearm under the mattress. The case was put to the jury on alternative theories of the defendant's actual or constructive possession of the firearm. On appeal, the defendant claims that the judge committed reversible error when he denied the defendant's motion for a required finding of not guilty because the evidence at the close of the Commonwealth's case was insufficient to convict him under either theory.
412 Mass. 267 , 272 (1992) ("A conviction may be properly based entirely on circumstantial evidence so long as that evidence establishes the defendant's guilt beyond a reasonable doubt").
The evidence presented at trial supported an inference that the defendant placed the firearm under the mattress. When first seen by the police, the defendant was attempting to enter 43 Harvard Street but was unable to open the door. When confronted by the police, he appeared "nervous" and "apprehensive." While talking to the police, the defendant placed a baseball batting glove on his right hand. Thereafter, when Ms. Lewis opened the previously locked door to the first-floor apartment and came out, the defendant pushed her to one side and ran into the apartment. [Note 2] The defendant immediately entered a bedroom, closed the door, and prevented Ms. Lewis and Officer Rooney from entering the room. He was seen bending down near the middle part of the bed in which the firearm was found. The firearm had not been under the mattress the day before the incident. Therefore, the evidence viewed in the light most favorable to the Commonwealth supports the inference that the defendant, at the time of the initial confrontation with the police, had in his possession the firearm and, afraid it would be discovered, panicked, ran into the bedroom, and hid it under the mattress.
The evidence was also sufficient as to constructive possession. " 'Constructive possession' requires proof that the defendant had 'knowledge coupled with the ability and intention to exercise dominion and control.' " Commonwealth v. Sann Than, 442 Mass. 748 , 751 (2004), quoting from Commonwealth v. Sespedes, 442 Mass. 95 , 99 (2004). Knowledge may be established by circumstantial evidence "if the evidence warrants a reasonable inference to that effect." Commonwealth v. Cotto, 69 Mass. App. Ct. 589 , 592 (2007), quoting from Commonwealth v. Garcia, 409 Mass. 675 , 686 (1991). Intent to exercise dominion and control can be inferred from the defendant's conduct. See Commonwealth v. Whitlock, 39 Mass. App. Ct. 514 , 519 (1995). "It is settled . . .
that the requisite knowledge, power and intent to exercise control over a firearm may be shown by 'presence, supplemented by other incriminating evidence.' " Commonwealth v. Valentin, 55 Mass. App. Ct. 667 , 671 (2002), quoting from Commonwealth v. Albano, 373 Mass. 132 , 134 (1977). Here, the presence of the defendant was supplemented by evidence that he ran to the bedroom, proceeded directly to the bed where the firearm was discovered, and attempted to prevent others from entering the room.
Therefore, we reject the defendant's claim that the evidence was insufficient to support his convictions of possession of a firearm and possession of ammunition.
3. Ineffective assistance of counsel. On appeal, the defendant claims that his trial counsel was constitutionally ineffective because of misstatements made in his closing argument. "The occasions when a court can resolve an ineffective assistance claim on direct appeal are exceptional, and our case law strongly disfavors raising ineffective assistance claims on direct appeal." Commonwealth v. Zinser, 446 Mass. 807 , 809 n.2 (2006). "The claim may nevertheless be resolved on direct appeal when 'the factual basis of the [ineffective assistance] claim appears undisputedly on the trial record.' " Commonwealth v. Livington, 70 Mass. App. Ct. 745 , 748 (2007), quoting from Commonwealth v. Diaz, 448 Mass. 286 , 289 (2007). We consider the defendant's claim under that "narrow exception." Commonwealth v. Lynch, 70 Mass. App. Ct. 22 , 29 n.6 (2007).
The standards on which the effectiveness of counsel will be evaluated are well known. "The defendant must show that counsel's performance 'f[ell] measurably below that which might be expected from an ordinarily fallible lawyer' and that, as a result, the defendant has been deprived of 'an otherwise available, substantial ground of defence.' " Commonwealth v. McCrae, 54 Mass. App. Ct. 27 , 29 (2002), quoting from Commonwealth v. Saferian, 366 Mass. 89 , 96 (1974). Here, the defendant argues that certain misstatements in defense counsel's closing argument deprived him "of an otherwise available, substantial ground of defence." Commonwealth v. Saferian, supra at 96.
the firearm under the mattress. Defense counsel destroyed that defense by misstating the evidence in his closing argument. It is undisputed that no one testified to having observed the defendant with the firearm, or to having seen him place it under the mattress. Rather, Ms. Lewis, a Commonwealth witness, testified that when she got to the bedroom, "I saw [the defendant] bending by the bed. I don't know what he was looking for, but he quickly got up. I turned around and went back outside." Later, when she was asked by the prosecutor, "And could you see if he was doing anything?" she responded, "No. I didn't see anything." During closing argument, however, defense counsel told the jury, "Ms. Lewis . . . tells you on the stand that she goes into the bedroom and sees [the defendant] kneeling by the bed and sees him put something under it and then gets out and comes out and gets the officer" (emphasis added).
Defense counsel's comment was a complete misstatement of Ms. Lewis's testimony and was prejudicial because it undermined the defense advanced at trial. [Note 3] See Commonwealth v. Triplett, 398 Mass. 561 , 569 (1986) ("Counsel's statements in his closing were tantamount to an admission of his client's guilt, . . . and left the client denuded of a defense" [quotations and citations omitted]). In such circumstances, a defendant is denied effective assistance of counsel. Further, the prejudicial effect of the error was compounded by the prosecutor, who told the jury in her closing argument that the defendant was observed "stashing [the firearm] by Ms. Lewis."
The Commonwealth argues that the judge's instructions to the jury cured any error caused by the closing arguments. The judge gave the standard instructions as to the purpose of counsels' closing arguments and that the jurors, not the lawyers, are the fact finders. The instructions, however, did not address the errors in specific terms. Commonwealth v. Beaudry, 445 Mass. 577 , 585 (2005).
The evidence was not overwhelming, the errors went to the heart of the case, and there were no curative instructions. Accordingly, the judgments of the Superior Court are reversed, the verdicts are set aside, and the case is remanded to the Superior Court for a new trial.
[Note 1] Seyon Marshall lived in the first-floor apartment in a room across the hall from a room occupied by Kamau Lewis. Kamau and Seyon are Ms. Lewis's grandsons. It was in Kamau's room that a gun was found.
[Note 2] Officer Rooney testified that the defendant "ran into" the apartment. Ms. Lewis testified that the defendant "walked" into the apartment, but did not run. Because we accept the evidence in the light most favorable to the Commonwealth, we take Officer Rooney's version. Defense counsel, however, in his closing argument also adopted Officer Rooney's version, an error we address later in the opinion.
[Note 3] Defense counsel further undermined the defense by stating three times during his closing argument that the defendant "ran" or had "run" into the apartment. Although there was a basis for those statements based on Officer Rooney's testimony, Ms. Lewis testified that the defendant "walked in. He didn't run." See note 2, supra. Defense counsel should not have assumed the correctness of the testimony less favorable to the defendant on the point. See Commonwealth v. Triplett, 398 Mass. 561 , 568-569 (1986).

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Controlled Substances. Evidence, Certificate of drug analysis. Constitutional Law, Confrontation of witnesses, Harmless error. Error, Harmless. Practice, Criminal, Confrontation of witnesses, Harmless error.
This court concluded that, at the trial of a criminal complaint charging the defendant with possession of cocaine with intent to distribute, possession of marijuana with intent to distribute, and commission of a drug offense within a school zone, the admission in evidence of certificates of drug analysis, without testimony from the analyst who created the certificates, in violation of the defendant's rights under the confrontation clause of the Sixth Amendment to the United States Constitution, was not harmless beyond a reasonable doubt, where the properly admitted evidence regarding the composition of the substances at issue was conclusory and largely equivocal and therefore not so powerful as to nullify the effect that the certificates might have had on the jury or on the verdicts. [381-384] SPINA, J., dissenting.
COMPLAINT received and sworn to in the Brockton Division of the District Court Department on May 13, 2004.
The case was tried before Paul J. McCallum, J.
Jennifer J. Cox for the defendant.
Debra S. Krupp, Committee for Public Counsel Services, for Committee for Public Counsel Services.
Todd C. Pomerleau, Danielle M. Wood, & Sarah M. Unger for Pomerleau Wood LLP.
Kathryn Hayne Barnwell for John Dessources.
saw the defendant arguing with another man. After seeing the approaching cruiser, the defendant walked toward a parked automobile and then sat in its driver's seat; another man was sitting in the passenger seat. The officers, together with Sergeant Paul Binnaca, who had just arrived at the scene, approached the vehicle. After the defendant refused to comply with orders to keep his hands visible, Officer Cruickshank opened the driver side door, "grabbed" the defendant's arm, and began to remove the defendant from the vehicle. At that point, both Officer Cruickshank and Sergeant Binnaca saw the defendant reach into his waistband, remove a plastic bag, and throw it over his shoulder into the back seat. Officer Cruickshank then pulled the defendant out of the automobile. After the officers had handcuffed the defendant, Sergeant Binnaca retrieved the bag and told Officer Cruickshank that there were narcotics in the vehicle.
At trial, evidence apart from the drug certificates pertaining to the chemical nature of the substances recovered from the automobile consisted in its entirety in the following testimony of Officer Cruickshank and Sergeant Binnaca. Officer Cruickshank, who had been a police officer for seven years, and through whom all three drug certificates were admitted in evidence, described the bag the defendant had thrown into the rear seat as containing "[e]ight pieces of crack cocaine" and six smaller bags of "a green leafy substance believed to be marijuana." He testified that he found a bag containing "22.5 grams of marijuana" in the vehicle's center console. He later referred to the substances as the "suspected marijuana and the crack cocaine," and as a "green leafy substance" and "a white, rock-like substance" that, in apparent reference to the drug certificates, were "found to be" marijuana and crack cocaine, respectively. See note 1, supra.
2. Discussion. The Commonwealth correctly recognizes that the submission in evidence of the three drug certificates without the trial testimony of the analysts who signed the drug certificates violated the defendant's right of confrontation as guaranteed by the Sixth Amendment to the United States Constitution. See Melendez-Diaz, supra. Although the defendant did not object at trial to the admission of the drug certificates, for the reasons explained in Vasquez, supra at 355-360, we must determine whether the error was harmless beyond a reasonable doubt. For the reasons stated below, under our established law, which we recently discussed at length in Commonwealth v. Tyree, 455 Mass. 676 , 700-704 (2010), and in Vasquez, supra at 360-368, we are not satisfied beyond a reasonable doubt that the drug certificates "did not have an effect on the jury and did not contribute to the jury's verdicts." Commonwealth v. Tyree, supra at 701.
As the Commonwealth has conceded, the drug certificates, introduced as part of the prosecution's prima facie case, provided, in the words of the assistant district attorney on appeal, the "most powerful evidence" that the substances at issue were "cocaine" and "marijuana," proof of which was necessary to establish that the defendant had committed the crimes of which he was charged. See Commonwealth v. Farley, 443 Mass. 740 , 745 (2005) (Commonwealth must prove "each" and "every" element of crime beyond reasonable doubt); Commonwealth v. McGilvery, 74 Mass. App. Ct. 508 , 511 (2009) (proof "that a substance is a particular drug" is element of drug prosecutions). The judge did not give the jury a standard instruction that drug certificates are prima facie evidence of the identity of the substances, but the drug certificates, which the jury could review, stated as much. See note 1, supra.
circumstantial evidence," Commonwealth v. Dawson, 399 Mass. 465 , 467 (1987), and in some circumstances not present here such evidence may be "so powerful" as to "nullify any effect" that the drug certificates "might have had on the jury or the verdict[s]." Commonwealth v. Tyree, supra at 704 n.44, quoting Commonwealth v. Dagraca, 447 Mass. 546 , 555 (2006). Here, however, the arresting police officers, neither of whom was qualified as an expert in or had specialized training or experience in narcotics identification, offered only conclusory, and largely equivocal, testimony regarding the composition of the substances. The officers certainly "did not articulate how their expertise permitted them to identify the substances" as cocaine and marijuana. Commonwealth v. Melendez-Diaz, 76 Mass. App. Ct. 229 , 233 (2010). Moreover, the judge did not make a finding that either officer's experience permitted him to offer an opinion that the substances were particular narcotics. See Commonwealth v. Dawson, supra (when police or drug-user witness testifies as to nature of substance, trial judge "will first have to make a finding" that witness's experience with drug would "permit him to give an opinion as to what drug a particular substance was"). See also Vasquez, supra at 365. There was no evidence that the officers detected any identifiable odors or recognized any other distinguishing characteristics of the substances beyond their appearance. See Commonwealth v. Dawson, supra ("it would be a rare case in which a witness's statement that a particular substance looked like a controlled substance would alone be sufficient to support a conviction"). [Note 5] Officer Cruickshank testified that he did not see any drug transaction occur, nor did he recall finding any money, cellular telephones, or pagers on the defendant. There was no mention of drugs in the report of the fight at the gasoline station to which the officers responded.
substances seized from the automobile were packaged in a manner consistent with drug distribution, as Detective Keating testified. See note 4, supra. Moreover, when confronted by police, in addition to attempting to discard a plastic bag containing some substances, the defendant was uncooperative when the officers ordered him to keep his hands visible and attempted to escape from the police cruiser after his arrest. See note 2, supra; Commonwealth v. Oeun Lam, 420 Mass. 615 , 617 (1995), and cases cited ("Evidence of escape or attempted escape is admissible to prove consciousness of guilt"). Such evidence did not, however, relieve the Commonwealth from proving beyond a reasonable doubt that the substances were, in fact, particular illegal narcotics, i.e., cocaine and marijuana. See Commonwealth v. Farley, supra; Commonwealth v. McGilvery, supra. Taken separately or together, the police officers' testimony is not "so powerful" as to "nullify any effect" that the drug certificates "might have had" on the jury or the verdicts. Commonwealth v. Tyree, supra.
that error on appeal, to contest the issue." (Emphasis in original.) This is not a case where the facts independent of the drug certificates overwhelmingly prove the nature of the substances recovered from the automobile. Contrast Commonwealth v. Doherty, 411 Mass. 95 , 102 (1991), cert. denied, 502 U.S. 1094 (1992), quoting Francis v. Franklin, 471 U.S. 307, 325-326 (1985) (erroneous intent instruction harmless beyond reasonable doubt where "principals' intent was not in dispute" because facts " 'overwhelmingly preclude[d]' the absence of an intent to kill" on part of principals).
3. Conclusion. The judgments are reversed, the verdicts are set aside, and the cases are remanded to the District Court for a new trial consistent with this decision.
SPINA, J. (dissenting in part and concurring in part) . I dissent and concur for the reasons previously stated in my separate opinion in Commonwealth v. Vasquez, ante 350, 374-377 (2010).
[Note 1] Each drug certificate was prepared as a notarized affidavit, signed by an assistant analyst at the State Laboratory Institute of the Department of Public Health. One drug certificate "certif[ied]" that the substance in eight plastic bags submitted to the State laboratory by a Brockton police detective was "Cocaine, a derivative of Coca leaves, as defined in Chapter 94C." The other two drug certificates "certif[ied]" that substances in other bags submitted by the police were "Marijuana, as defined in Chapter 94C." The drug certificates also bore the following legend: "When properly executed, [this certificate] shall be prima facie evidence of the composition, quality, and the net weight of the narcotic or other drug, poison, medicine, or chemical analyzed . . ." (emphasis added).
[Note 2] The defendant was also convicted of resisting arrest, G. L. c. 268, § 32B, but this conviction is not before us.
[Note 3] We acknowledge the amicus briefs filed on behalf of the defendant by the Committee for Public Counsel Services, Pomerleau Wood LLP, and John Dessources.
[Note 4] An additional witness, Detective Thomas Keating, who was not involved in the arrest or investigation, testified that, on the basis of hypothetical questions describing what the officers had seized, the quantity and packaging style of the alleged narcotics suggested distribution. He did not evaluate the substances recovered from the vehicle or offer any testimony as to their chemical composition.
[Note 5] Protocols for the analysis of cocaine, heroin, and marijuana samples promulgated by the State police require that a chemical analysis be conducted for each substance; identification of the substance is not authorized on the basis of a visual inspection alone.